RAPID DETECTION AND QUANTIFICATION OF Vibrio parahaemolyticus IN COCKLES
(Anadara granosa) BY USING REAL-TIME PCR ~
Patricia Rowena Anak Mark Baran Saging
QR 81 S6 P314 1012
Bachelor of Science with Honours (Resource Biotechnology)
2012
ampR gl
~ ~
nlt 0 2
Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SARAWAK
Rapid detection and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
PKHIDMAT MAKLUMAT AKADEMIK
PATRICIA ROWENA ANAKMARK BARAN SAGING (24823)
A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours
(Resource Biotechnology)
Faculty of Resource Science and Technology UNIVERSITY MALAYSIA SARAW AK
2012
ACKNOWLEDGEMENTS
First and foremost I would like to thank God the Almighty for listening to my
prayers upon the completion of this Final Year Project A greatest appreciation and heartfelt
gratitude also goes to my respected supervisor Dr Lesley Maurice Bilung and to my coshy
supervisor Dr Micky Vincent for their endless guidance support and advice throughout
conducting this project
Furthermore I dedicate many thanks to the post-graduate students from
Microbiology Laboratory Velnetti Linang Christy and Zena for their help concern and
suggestion on laboratory work until final accomplishment of this project Besides that I
would like to thank Mr Azis who has assisted in term of technical and material matters Lest
to forget this acknowledgment is also expressed especially to my laboratory mates Sherry
Sim Nur Quraitu Aini Aimi Syazana Dellroy Marlina and Miza for their guidance as well
as for making this project filled with positive atmosphere
Last but not least special thanks to my family and friends for their endless moral
supports and for their word full of compliments throughout -thiS Final Year Project and thesis
preparation
DECLARATION
I hereby declare that the study entitled Rapid detection and quantification of V
parahaemolyticus in cockles (Anadara granosa) by using Real-Time peR is my original
work and that all the sources that I have quoted and referred to have been acknowledged by
means of complete references It has been submitted and shall not be submitted in any form
to any institution or other university
Students signature Date
II
- -~ ~ -------~
PUSlt Khidmlt Maldumat Akademik UNlVERSm MALAVSIA SARAWAK
T ABLE OF CONTENTS
Page
ACKNOWLEDGEMENT I
DECLARATION II
LIST OF TABLES
III
VI
VIII
LIST OF FIGURES VII
TABLE OF CONTENTS
LIST OF ABBREVIATIONS
XABSTRACT
XABSTRAK
CHAPTER 1 INTRODUCTION
CHAPTER 2 LITERA TURE REVIEWS 3
21 Vibrio Genus 3
22 Vibrio parahaemolyticus 3
23 Cockles 4
24 Route of Transmission of V par~haemolyticus 5
525 Clinical Manifestation of V parahaemolyticus
626 Virulence Properties of V parahaemolyticus
727 Isolation and Identification
728 Real-Time Polymerase Chain Reaction (RT-PCR)
9CHAPTER 3 MA TERIALS AND METHODS
31 Sample Collection 9
32 Sample Preparation and Enrichment of Bacterial 9 Sample
1033 Genomic DNA Preparation
III
J
34 Selective Isolation and Detection of 10 V parahaemolyticus
35 Polymerase Chain Reaction (PCR) 11
36 Agarose Gel Electrophoresis 12
37 Sensitivity Study with Pure Culture for Standard 12 Curve
371 Dilution and Bacterial cell count 12
372 Bacterial cell count by Hemocytometer l3
14373 Spiking of Pure Culture into Raw Fish
1438 Specificity Study using Specific PCR
1539 Real-Time PCR Assay
40 RESULTS
1741 Bacterial Identification
1942 Sensitivity Studies of Pure Culture of V parahaemolyticus
421 Specific PCR Analysis Targeting tlh-gene bull 419 from Tilapia fish prior u~e for Spiking
Purpose - ~
20422 Vibrio parahaemolyticus Cell Count by Hemocytometer
423 Spiking and Dilution Samples for Standard 21 Curve
43 Specificity Study using Specific PCR 23
44 RT-PCR Assay for Detection of tlh positive 24 V parahaemolyticus in Cockles Samples
2650 DISCUSSION
60 CONCLUSION 30
REFERENCES 31
IV
35 APPENDICES
-
v
LIST OF TABLES
Table 1 PCR parameters
Table 2 The mixture was subjected to 35 cycles on a thermo cycler (Eppendorfreg Mastercycle Personal)
Table 3 Bacterial strains for specific PCR
Table 4 Cycling conditions ofRT-PCR of 40 cycles on Rotor Gene 6000 Corbett (Australia)
Table 5 RT-PCR reagent component
Table 6 Raw data for spiking samples with standard deviation value for standard curve
Table 7 Raw data for initial concentration for spiking samples
Table 8 Raw data for serial dilution samples with standard deviation value for standard curve
Page
11
12
15
15
16
35
35
36
VI
--------
LIST OF FIGURES
Page
Figure I The green colonies of V parahaemolyticus isolates on TCBS 18 agar plate
Figure 2 The mauve colonies of V parahaemolyticus isolates on 18 CHROMagartrade Vibrio
Figure 3 Agarose gel electrophoresis of the specific PCR pattern for 19 detection of Tilapia fish (Oreochromis niloticus) sample used prior to spiking using primers MV2B-tl which is tlhshybased gene targeting thennolabile hemolysin (tlh) gene Lanes L-l kbp DNA ladder (Invitrogen) I-Positive control (V parahemolyticus) 2-Tilapia fish sample 3-Non-template control
Figure 4 Hemocytometer count of undiluted V parahaemolyticus 20 culture viewed under 400x magnification
Figure 5 Standard curves for the number of V parahaemolyticus cells 21 versus Ct values of spiking samples The error bars indicate the standard deviations in two independent experiments
Figure 6 Standard curves for the number of V parahaemolyticus cells 22 versus Ct values of serial dilution samples The error bars indicate the standard deviations in two independent experiments
Figure 7 Agarose gel electrophoresis of the specific PCR pattern for 23 specificity test of MV2B-tl primer with other bactlrial strains Lanes L-l kbp DNA ladder (Invitrogen) I-Non-template control 2-Positive control (V parahaemolyticus) 3-Cockle sample 4-Fish sample 5-Escherichia coli (A TCC 56021) 6shyEscherichia coli KOll (ATCC 55124) 7-Saccharomyces cerevisiae (A TCC 24859) 8- Cryptococcus curvatus (ATCC 20509) 9- Gloephyllum trabeum (A TCC 11539) 10shyPhanerochaete chrysosporium (ATCC 24725) 11shySalmonella flexneri (ATCC 12022)
Figure 8 Melting curve analysis from 3 cockle samples with resulted 24 number of fluorescence against temperature
Figure 9 Quantification analysis from 3 cockle samples with resulted 25 number of cycle versus the number of fluorescence units for each sample used to determine Ct
VII
LIST OF ABBREVIATIONS
APW Alkaline Peptone Water
ATCC American Type Culture Collection
em centimetre
Ct Threshold cycle
DNA Deoxyribonucleic acid
dNTPs deoxynucleoside triphosphates
EDTA Ethylenediamine tetra-acetic acid
epsM extracellular secretion protein
EtBr Ethidium bromide
g gram
kbp kilo base pair
LB Luria Bertani
m meter
MgCh magnesium chloride
min minute(s)
mL mililiter
mM milimolar
NaCI sodium chloride
run nanometer
PCR Polymerase Chain Reaction
pH potential hydrogen
(shy correlation coefficient
rpm revoluhon per minute
VIII
RT-PCR
sec
TBE
TCBS
tdh
tlh
trh
UV
IJIll
~
middotC
Real-Time Polymerase Chain Reaction
second(s)
Tris-Borate-EDTA
Thiosulphate-Citrate-Bile Salts-Sucrose
thermostable direct hemolysin
thermolabile hemolysin
thermostable direct hemolysin related hemolysin
ultraviolet
micrometer
microliter
degree celsius
percentage
bull
IX
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
ampR gl
~ ~
nlt 0 2
Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SARAWAK
Rapid detection and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
PKHIDMAT MAKLUMAT AKADEMIK
PATRICIA ROWENA ANAKMARK BARAN SAGING (24823)
A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours
(Resource Biotechnology)
Faculty of Resource Science and Technology UNIVERSITY MALAYSIA SARAW AK
2012
ACKNOWLEDGEMENTS
First and foremost I would like to thank God the Almighty for listening to my
prayers upon the completion of this Final Year Project A greatest appreciation and heartfelt
gratitude also goes to my respected supervisor Dr Lesley Maurice Bilung and to my coshy
supervisor Dr Micky Vincent for their endless guidance support and advice throughout
conducting this project
Furthermore I dedicate many thanks to the post-graduate students from
Microbiology Laboratory Velnetti Linang Christy and Zena for their help concern and
suggestion on laboratory work until final accomplishment of this project Besides that I
would like to thank Mr Azis who has assisted in term of technical and material matters Lest
to forget this acknowledgment is also expressed especially to my laboratory mates Sherry
Sim Nur Quraitu Aini Aimi Syazana Dellroy Marlina and Miza for their guidance as well
as for making this project filled with positive atmosphere
Last but not least special thanks to my family and friends for their endless moral
supports and for their word full of compliments throughout -thiS Final Year Project and thesis
preparation
DECLARATION
I hereby declare that the study entitled Rapid detection and quantification of V
parahaemolyticus in cockles (Anadara granosa) by using Real-Time peR is my original
work and that all the sources that I have quoted and referred to have been acknowledged by
means of complete references It has been submitted and shall not be submitted in any form
to any institution or other university
Students signature Date
II
- -~ ~ -------~
PUSlt Khidmlt Maldumat Akademik UNlVERSm MALAVSIA SARAWAK
T ABLE OF CONTENTS
Page
ACKNOWLEDGEMENT I
DECLARATION II
LIST OF TABLES
III
VI
VIII
LIST OF FIGURES VII
TABLE OF CONTENTS
LIST OF ABBREVIATIONS
XABSTRACT
XABSTRAK
CHAPTER 1 INTRODUCTION
CHAPTER 2 LITERA TURE REVIEWS 3
21 Vibrio Genus 3
22 Vibrio parahaemolyticus 3
23 Cockles 4
24 Route of Transmission of V par~haemolyticus 5
525 Clinical Manifestation of V parahaemolyticus
626 Virulence Properties of V parahaemolyticus
727 Isolation and Identification
728 Real-Time Polymerase Chain Reaction (RT-PCR)
9CHAPTER 3 MA TERIALS AND METHODS
31 Sample Collection 9
32 Sample Preparation and Enrichment of Bacterial 9 Sample
1033 Genomic DNA Preparation
III
J
34 Selective Isolation and Detection of 10 V parahaemolyticus
35 Polymerase Chain Reaction (PCR) 11
36 Agarose Gel Electrophoresis 12
37 Sensitivity Study with Pure Culture for Standard 12 Curve
371 Dilution and Bacterial cell count 12
372 Bacterial cell count by Hemocytometer l3
14373 Spiking of Pure Culture into Raw Fish
1438 Specificity Study using Specific PCR
1539 Real-Time PCR Assay
40 RESULTS
1741 Bacterial Identification
1942 Sensitivity Studies of Pure Culture of V parahaemolyticus
421 Specific PCR Analysis Targeting tlh-gene bull 419 from Tilapia fish prior u~e for Spiking
Purpose - ~
20422 Vibrio parahaemolyticus Cell Count by Hemocytometer
423 Spiking and Dilution Samples for Standard 21 Curve
43 Specificity Study using Specific PCR 23
44 RT-PCR Assay for Detection of tlh positive 24 V parahaemolyticus in Cockles Samples
2650 DISCUSSION
60 CONCLUSION 30
REFERENCES 31
IV
35 APPENDICES
-
v
LIST OF TABLES
Table 1 PCR parameters
Table 2 The mixture was subjected to 35 cycles on a thermo cycler (Eppendorfreg Mastercycle Personal)
Table 3 Bacterial strains for specific PCR
Table 4 Cycling conditions ofRT-PCR of 40 cycles on Rotor Gene 6000 Corbett (Australia)
Table 5 RT-PCR reagent component
Table 6 Raw data for spiking samples with standard deviation value for standard curve
Table 7 Raw data for initial concentration for spiking samples
Table 8 Raw data for serial dilution samples with standard deviation value for standard curve
Page
11
12
15
15
16
35
35
36
VI
--------
LIST OF FIGURES
Page
Figure I The green colonies of V parahaemolyticus isolates on TCBS 18 agar plate
Figure 2 The mauve colonies of V parahaemolyticus isolates on 18 CHROMagartrade Vibrio
Figure 3 Agarose gel electrophoresis of the specific PCR pattern for 19 detection of Tilapia fish (Oreochromis niloticus) sample used prior to spiking using primers MV2B-tl which is tlhshybased gene targeting thennolabile hemolysin (tlh) gene Lanes L-l kbp DNA ladder (Invitrogen) I-Positive control (V parahemolyticus) 2-Tilapia fish sample 3-Non-template control
Figure 4 Hemocytometer count of undiluted V parahaemolyticus 20 culture viewed under 400x magnification
Figure 5 Standard curves for the number of V parahaemolyticus cells 21 versus Ct values of spiking samples The error bars indicate the standard deviations in two independent experiments
Figure 6 Standard curves for the number of V parahaemolyticus cells 22 versus Ct values of serial dilution samples The error bars indicate the standard deviations in two independent experiments
Figure 7 Agarose gel electrophoresis of the specific PCR pattern for 23 specificity test of MV2B-tl primer with other bactlrial strains Lanes L-l kbp DNA ladder (Invitrogen) I-Non-template control 2-Positive control (V parahaemolyticus) 3-Cockle sample 4-Fish sample 5-Escherichia coli (A TCC 56021) 6shyEscherichia coli KOll (ATCC 55124) 7-Saccharomyces cerevisiae (A TCC 24859) 8- Cryptococcus curvatus (ATCC 20509) 9- Gloephyllum trabeum (A TCC 11539) 10shyPhanerochaete chrysosporium (ATCC 24725) 11shySalmonella flexneri (ATCC 12022)
Figure 8 Melting curve analysis from 3 cockle samples with resulted 24 number of fluorescence against temperature
Figure 9 Quantification analysis from 3 cockle samples with resulted 25 number of cycle versus the number of fluorescence units for each sample used to determine Ct
VII
LIST OF ABBREVIATIONS
APW Alkaline Peptone Water
ATCC American Type Culture Collection
em centimetre
Ct Threshold cycle
DNA Deoxyribonucleic acid
dNTPs deoxynucleoside triphosphates
EDTA Ethylenediamine tetra-acetic acid
epsM extracellular secretion protein
EtBr Ethidium bromide
g gram
kbp kilo base pair
LB Luria Bertani
m meter
MgCh magnesium chloride
min minute(s)
mL mililiter
mM milimolar
NaCI sodium chloride
run nanometer
PCR Polymerase Chain Reaction
pH potential hydrogen
(shy correlation coefficient
rpm revoluhon per minute
VIII
RT-PCR
sec
TBE
TCBS
tdh
tlh
trh
UV
IJIll
~
middotC
Real-Time Polymerase Chain Reaction
second(s)
Tris-Borate-EDTA
Thiosulphate-Citrate-Bile Salts-Sucrose
thermostable direct hemolysin
thermolabile hemolysin
thermostable direct hemolysin related hemolysin
ultraviolet
micrometer
microliter
degree celsius
percentage
bull
IX
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
ACKNOWLEDGEMENTS
First and foremost I would like to thank God the Almighty for listening to my
prayers upon the completion of this Final Year Project A greatest appreciation and heartfelt
gratitude also goes to my respected supervisor Dr Lesley Maurice Bilung and to my coshy
supervisor Dr Micky Vincent for their endless guidance support and advice throughout
conducting this project
Furthermore I dedicate many thanks to the post-graduate students from
Microbiology Laboratory Velnetti Linang Christy and Zena for their help concern and
suggestion on laboratory work until final accomplishment of this project Besides that I
would like to thank Mr Azis who has assisted in term of technical and material matters Lest
to forget this acknowledgment is also expressed especially to my laboratory mates Sherry
Sim Nur Quraitu Aini Aimi Syazana Dellroy Marlina and Miza for their guidance as well
as for making this project filled with positive atmosphere
Last but not least special thanks to my family and friends for their endless moral
supports and for their word full of compliments throughout -thiS Final Year Project and thesis
preparation
DECLARATION
I hereby declare that the study entitled Rapid detection and quantification of V
parahaemolyticus in cockles (Anadara granosa) by using Real-Time peR is my original
work and that all the sources that I have quoted and referred to have been acknowledged by
means of complete references It has been submitted and shall not be submitted in any form
to any institution or other university
Students signature Date
II
- -~ ~ -------~
PUSlt Khidmlt Maldumat Akademik UNlVERSm MALAVSIA SARAWAK
T ABLE OF CONTENTS
Page
ACKNOWLEDGEMENT I
DECLARATION II
LIST OF TABLES
III
VI
VIII
LIST OF FIGURES VII
TABLE OF CONTENTS
LIST OF ABBREVIATIONS
XABSTRACT
XABSTRAK
CHAPTER 1 INTRODUCTION
CHAPTER 2 LITERA TURE REVIEWS 3
21 Vibrio Genus 3
22 Vibrio parahaemolyticus 3
23 Cockles 4
24 Route of Transmission of V par~haemolyticus 5
525 Clinical Manifestation of V parahaemolyticus
626 Virulence Properties of V parahaemolyticus
727 Isolation and Identification
728 Real-Time Polymerase Chain Reaction (RT-PCR)
9CHAPTER 3 MA TERIALS AND METHODS
31 Sample Collection 9
32 Sample Preparation and Enrichment of Bacterial 9 Sample
1033 Genomic DNA Preparation
III
J
34 Selective Isolation and Detection of 10 V parahaemolyticus
35 Polymerase Chain Reaction (PCR) 11
36 Agarose Gel Electrophoresis 12
37 Sensitivity Study with Pure Culture for Standard 12 Curve
371 Dilution and Bacterial cell count 12
372 Bacterial cell count by Hemocytometer l3
14373 Spiking of Pure Culture into Raw Fish
1438 Specificity Study using Specific PCR
1539 Real-Time PCR Assay
40 RESULTS
1741 Bacterial Identification
1942 Sensitivity Studies of Pure Culture of V parahaemolyticus
421 Specific PCR Analysis Targeting tlh-gene bull 419 from Tilapia fish prior u~e for Spiking
Purpose - ~
20422 Vibrio parahaemolyticus Cell Count by Hemocytometer
423 Spiking and Dilution Samples for Standard 21 Curve
43 Specificity Study using Specific PCR 23
44 RT-PCR Assay for Detection of tlh positive 24 V parahaemolyticus in Cockles Samples
2650 DISCUSSION
60 CONCLUSION 30
REFERENCES 31
IV
35 APPENDICES
-
v
LIST OF TABLES
Table 1 PCR parameters
Table 2 The mixture was subjected to 35 cycles on a thermo cycler (Eppendorfreg Mastercycle Personal)
Table 3 Bacterial strains for specific PCR
Table 4 Cycling conditions ofRT-PCR of 40 cycles on Rotor Gene 6000 Corbett (Australia)
Table 5 RT-PCR reagent component
Table 6 Raw data for spiking samples with standard deviation value for standard curve
Table 7 Raw data for initial concentration for spiking samples
Table 8 Raw data for serial dilution samples with standard deviation value for standard curve
Page
11
12
15
15
16
35
35
36
VI
--------
LIST OF FIGURES
Page
Figure I The green colonies of V parahaemolyticus isolates on TCBS 18 agar plate
Figure 2 The mauve colonies of V parahaemolyticus isolates on 18 CHROMagartrade Vibrio
Figure 3 Agarose gel electrophoresis of the specific PCR pattern for 19 detection of Tilapia fish (Oreochromis niloticus) sample used prior to spiking using primers MV2B-tl which is tlhshybased gene targeting thennolabile hemolysin (tlh) gene Lanes L-l kbp DNA ladder (Invitrogen) I-Positive control (V parahemolyticus) 2-Tilapia fish sample 3-Non-template control
Figure 4 Hemocytometer count of undiluted V parahaemolyticus 20 culture viewed under 400x magnification
Figure 5 Standard curves for the number of V parahaemolyticus cells 21 versus Ct values of spiking samples The error bars indicate the standard deviations in two independent experiments
Figure 6 Standard curves for the number of V parahaemolyticus cells 22 versus Ct values of serial dilution samples The error bars indicate the standard deviations in two independent experiments
Figure 7 Agarose gel electrophoresis of the specific PCR pattern for 23 specificity test of MV2B-tl primer with other bactlrial strains Lanes L-l kbp DNA ladder (Invitrogen) I-Non-template control 2-Positive control (V parahaemolyticus) 3-Cockle sample 4-Fish sample 5-Escherichia coli (A TCC 56021) 6shyEscherichia coli KOll (ATCC 55124) 7-Saccharomyces cerevisiae (A TCC 24859) 8- Cryptococcus curvatus (ATCC 20509) 9- Gloephyllum trabeum (A TCC 11539) 10shyPhanerochaete chrysosporium (ATCC 24725) 11shySalmonella flexneri (ATCC 12022)
Figure 8 Melting curve analysis from 3 cockle samples with resulted 24 number of fluorescence against temperature
Figure 9 Quantification analysis from 3 cockle samples with resulted 25 number of cycle versus the number of fluorescence units for each sample used to determine Ct
VII
LIST OF ABBREVIATIONS
APW Alkaline Peptone Water
ATCC American Type Culture Collection
em centimetre
Ct Threshold cycle
DNA Deoxyribonucleic acid
dNTPs deoxynucleoside triphosphates
EDTA Ethylenediamine tetra-acetic acid
epsM extracellular secretion protein
EtBr Ethidium bromide
g gram
kbp kilo base pair
LB Luria Bertani
m meter
MgCh magnesium chloride
min minute(s)
mL mililiter
mM milimolar
NaCI sodium chloride
run nanometer
PCR Polymerase Chain Reaction
pH potential hydrogen
(shy correlation coefficient
rpm revoluhon per minute
VIII
RT-PCR
sec
TBE
TCBS
tdh
tlh
trh
UV
IJIll
~
middotC
Real-Time Polymerase Chain Reaction
second(s)
Tris-Borate-EDTA
Thiosulphate-Citrate-Bile Salts-Sucrose
thermostable direct hemolysin
thermolabile hemolysin
thermostable direct hemolysin related hemolysin
ultraviolet
micrometer
microliter
degree celsius
percentage
bull
IX
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
DECLARATION
I hereby declare that the study entitled Rapid detection and quantification of V
parahaemolyticus in cockles (Anadara granosa) by using Real-Time peR is my original
work and that all the sources that I have quoted and referred to have been acknowledged by
means of complete references It has been submitted and shall not be submitted in any form
to any institution or other university
Students signature Date
II
- -~ ~ -------~
PUSlt Khidmlt Maldumat Akademik UNlVERSm MALAVSIA SARAWAK
T ABLE OF CONTENTS
Page
ACKNOWLEDGEMENT I
DECLARATION II
LIST OF TABLES
III
VI
VIII
LIST OF FIGURES VII
TABLE OF CONTENTS
LIST OF ABBREVIATIONS
XABSTRACT
XABSTRAK
CHAPTER 1 INTRODUCTION
CHAPTER 2 LITERA TURE REVIEWS 3
21 Vibrio Genus 3
22 Vibrio parahaemolyticus 3
23 Cockles 4
24 Route of Transmission of V par~haemolyticus 5
525 Clinical Manifestation of V parahaemolyticus
626 Virulence Properties of V parahaemolyticus
727 Isolation and Identification
728 Real-Time Polymerase Chain Reaction (RT-PCR)
9CHAPTER 3 MA TERIALS AND METHODS
31 Sample Collection 9
32 Sample Preparation and Enrichment of Bacterial 9 Sample
1033 Genomic DNA Preparation
III
J
34 Selective Isolation and Detection of 10 V parahaemolyticus
35 Polymerase Chain Reaction (PCR) 11
36 Agarose Gel Electrophoresis 12
37 Sensitivity Study with Pure Culture for Standard 12 Curve
371 Dilution and Bacterial cell count 12
372 Bacterial cell count by Hemocytometer l3
14373 Spiking of Pure Culture into Raw Fish
1438 Specificity Study using Specific PCR
1539 Real-Time PCR Assay
40 RESULTS
1741 Bacterial Identification
1942 Sensitivity Studies of Pure Culture of V parahaemolyticus
421 Specific PCR Analysis Targeting tlh-gene bull 419 from Tilapia fish prior u~e for Spiking
Purpose - ~
20422 Vibrio parahaemolyticus Cell Count by Hemocytometer
423 Spiking and Dilution Samples for Standard 21 Curve
43 Specificity Study using Specific PCR 23
44 RT-PCR Assay for Detection of tlh positive 24 V parahaemolyticus in Cockles Samples
2650 DISCUSSION
60 CONCLUSION 30
REFERENCES 31
IV
35 APPENDICES
-
v
LIST OF TABLES
Table 1 PCR parameters
Table 2 The mixture was subjected to 35 cycles on a thermo cycler (Eppendorfreg Mastercycle Personal)
Table 3 Bacterial strains for specific PCR
Table 4 Cycling conditions ofRT-PCR of 40 cycles on Rotor Gene 6000 Corbett (Australia)
Table 5 RT-PCR reagent component
Table 6 Raw data for spiking samples with standard deviation value for standard curve
Table 7 Raw data for initial concentration for spiking samples
Table 8 Raw data for serial dilution samples with standard deviation value for standard curve
Page
11
12
15
15
16
35
35
36
VI
--------
LIST OF FIGURES
Page
Figure I The green colonies of V parahaemolyticus isolates on TCBS 18 agar plate
Figure 2 The mauve colonies of V parahaemolyticus isolates on 18 CHROMagartrade Vibrio
Figure 3 Agarose gel electrophoresis of the specific PCR pattern for 19 detection of Tilapia fish (Oreochromis niloticus) sample used prior to spiking using primers MV2B-tl which is tlhshybased gene targeting thennolabile hemolysin (tlh) gene Lanes L-l kbp DNA ladder (Invitrogen) I-Positive control (V parahemolyticus) 2-Tilapia fish sample 3-Non-template control
Figure 4 Hemocytometer count of undiluted V parahaemolyticus 20 culture viewed under 400x magnification
Figure 5 Standard curves for the number of V parahaemolyticus cells 21 versus Ct values of spiking samples The error bars indicate the standard deviations in two independent experiments
Figure 6 Standard curves for the number of V parahaemolyticus cells 22 versus Ct values of serial dilution samples The error bars indicate the standard deviations in two independent experiments
Figure 7 Agarose gel electrophoresis of the specific PCR pattern for 23 specificity test of MV2B-tl primer with other bactlrial strains Lanes L-l kbp DNA ladder (Invitrogen) I-Non-template control 2-Positive control (V parahaemolyticus) 3-Cockle sample 4-Fish sample 5-Escherichia coli (A TCC 56021) 6shyEscherichia coli KOll (ATCC 55124) 7-Saccharomyces cerevisiae (A TCC 24859) 8- Cryptococcus curvatus (ATCC 20509) 9- Gloephyllum trabeum (A TCC 11539) 10shyPhanerochaete chrysosporium (ATCC 24725) 11shySalmonella flexneri (ATCC 12022)
Figure 8 Melting curve analysis from 3 cockle samples with resulted 24 number of fluorescence against temperature
Figure 9 Quantification analysis from 3 cockle samples with resulted 25 number of cycle versus the number of fluorescence units for each sample used to determine Ct
VII
LIST OF ABBREVIATIONS
APW Alkaline Peptone Water
ATCC American Type Culture Collection
em centimetre
Ct Threshold cycle
DNA Deoxyribonucleic acid
dNTPs deoxynucleoside triphosphates
EDTA Ethylenediamine tetra-acetic acid
epsM extracellular secretion protein
EtBr Ethidium bromide
g gram
kbp kilo base pair
LB Luria Bertani
m meter
MgCh magnesium chloride
min minute(s)
mL mililiter
mM milimolar
NaCI sodium chloride
run nanometer
PCR Polymerase Chain Reaction
pH potential hydrogen
(shy correlation coefficient
rpm revoluhon per minute
VIII
RT-PCR
sec
TBE
TCBS
tdh
tlh
trh
UV
IJIll
~
middotC
Real-Time Polymerase Chain Reaction
second(s)
Tris-Borate-EDTA
Thiosulphate-Citrate-Bile Salts-Sucrose
thermostable direct hemolysin
thermolabile hemolysin
thermostable direct hemolysin related hemolysin
ultraviolet
micrometer
microliter
degree celsius
percentage
bull
IX
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
- -~ ~ -------~
PUSlt Khidmlt Maldumat Akademik UNlVERSm MALAVSIA SARAWAK
T ABLE OF CONTENTS
Page
ACKNOWLEDGEMENT I
DECLARATION II
LIST OF TABLES
III
VI
VIII
LIST OF FIGURES VII
TABLE OF CONTENTS
LIST OF ABBREVIATIONS
XABSTRACT
XABSTRAK
CHAPTER 1 INTRODUCTION
CHAPTER 2 LITERA TURE REVIEWS 3
21 Vibrio Genus 3
22 Vibrio parahaemolyticus 3
23 Cockles 4
24 Route of Transmission of V par~haemolyticus 5
525 Clinical Manifestation of V parahaemolyticus
626 Virulence Properties of V parahaemolyticus
727 Isolation and Identification
728 Real-Time Polymerase Chain Reaction (RT-PCR)
9CHAPTER 3 MA TERIALS AND METHODS
31 Sample Collection 9
32 Sample Preparation and Enrichment of Bacterial 9 Sample
1033 Genomic DNA Preparation
III
J
34 Selective Isolation and Detection of 10 V parahaemolyticus
35 Polymerase Chain Reaction (PCR) 11
36 Agarose Gel Electrophoresis 12
37 Sensitivity Study with Pure Culture for Standard 12 Curve
371 Dilution and Bacterial cell count 12
372 Bacterial cell count by Hemocytometer l3
14373 Spiking of Pure Culture into Raw Fish
1438 Specificity Study using Specific PCR
1539 Real-Time PCR Assay
40 RESULTS
1741 Bacterial Identification
1942 Sensitivity Studies of Pure Culture of V parahaemolyticus
421 Specific PCR Analysis Targeting tlh-gene bull 419 from Tilapia fish prior u~e for Spiking
Purpose - ~
20422 Vibrio parahaemolyticus Cell Count by Hemocytometer
423 Spiking and Dilution Samples for Standard 21 Curve
43 Specificity Study using Specific PCR 23
44 RT-PCR Assay for Detection of tlh positive 24 V parahaemolyticus in Cockles Samples
2650 DISCUSSION
60 CONCLUSION 30
REFERENCES 31
IV
35 APPENDICES
-
v
LIST OF TABLES
Table 1 PCR parameters
Table 2 The mixture was subjected to 35 cycles on a thermo cycler (Eppendorfreg Mastercycle Personal)
Table 3 Bacterial strains for specific PCR
Table 4 Cycling conditions ofRT-PCR of 40 cycles on Rotor Gene 6000 Corbett (Australia)
Table 5 RT-PCR reagent component
Table 6 Raw data for spiking samples with standard deviation value for standard curve
Table 7 Raw data for initial concentration for spiking samples
Table 8 Raw data for serial dilution samples with standard deviation value for standard curve
Page
11
12
15
15
16
35
35
36
VI
--------
LIST OF FIGURES
Page
Figure I The green colonies of V parahaemolyticus isolates on TCBS 18 agar plate
Figure 2 The mauve colonies of V parahaemolyticus isolates on 18 CHROMagartrade Vibrio
Figure 3 Agarose gel electrophoresis of the specific PCR pattern for 19 detection of Tilapia fish (Oreochromis niloticus) sample used prior to spiking using primers MV2B-tl which is tlhshybased gene targeting thennolabile hemolysin (tlh) gene Lanes L-l kbp DNA ladder (Invitrogen) I-Positive control (V parahemolyticus) 2-Tilapia fish sample 3-Non-template control
Figure 4 Hemocytometer count of undiluted V parahaemolyticus 20 culture viewed under 400x magnification
Figure 5 Standard curves for the number of V parahaemolyticus cells 21 versus Ct values of spiking samples The error bars indicate the standard deviations in two independent experiments
Figure 6 Standard curves for the number of V parahaemolyticus cells 22 versus Ct values of serial dilution samples The error bars indicate the standard deviations in two independent experiments
Figure 7 Agarose gel electrophoresis of the specific PCR pattern for 23 specificity test of MV2B-tl primer with other bactlrial strains Lanes L-l kbp DNA ladder (Invitrogen) I-Non-template control 2-Positive control (V parahaemolyticus) 3-Cockle sample 4-Fish sample 5-Escherichia coli (A TCC 56021) 6shyEscherichia coli KOll (ATCC 55124) 7-Saccharomyces cerevisiae (A TCC 24859) 8- Cryptococcus curvatus (ATCC 20509) 9- Gloephyllum trabeum (A TCC 11539) 10shyPhanerochaete chrysosporium (ATCC 24725) 11shySalmonella flexneri (ATCC 12022)
Figure 8 Melting curve analysis from 3 cockle samples with resulted 24 number of fluorescence against temperature
Figure 9 Quantification analysis from 3 cockle samples with resulted 25 number of cycle versus the number of fluorescence units for each sample used to determine Ct
VII
LIST OF ABBREVIATIONS
APW Alkaline Peptone Water
ATCC American Type Culture Collection
em centimetre
Ct Threshold cycle
DNA Deoxyribonucleic acid
dNTPs deoxynucleoside triphosphates
EDTA Ethylenediamine tetra-acetic acid
epsM extracellular secretion protein
EtBr Ethidium bromide
g gram
kbp kilo base pair
LB Luria Bertani
m meter
MgCh magnesium chloride
min minute(s)
mL mililiter
mM milimolar
NaCI sodium chloride
run nanometer
PCR Polymerase Chain Reaction
pH potential hydrogen
(shy correlation coefficient
rpm revoluhon per minute
VIII
RT-PCR
sec
TBE
TCBS
tdh
tlh
trh
UV
IJIll
~
middotC
Real-Time Polymerase Chain Reaction
second(s)
Tris-Borate-EDTA
Thiosulphate-Citrate-Bile Salts-Sucrose
thermostable direct hemolysin
thermolabile hemolysin
thermostable direct hemolysin related hemolysin
ultraviolet
micrometer
microliter
degree celsius
percentage
bull
IX
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
J
34 Selective Isolation and Detection of 10 V parahaemolyticus
35 Polymerase Chain Reaction (PCR) 11
36 Agarose Gel Electrophoresis 12
37 Sensitivity Study with Pure Culture for Standard 12 Curve
371 Dilution and Bacterial cell count 12
372 Bacterial cell count by Hemocytometer l3
14373 Spiking of Pure Culture into Raw Fish
1438 Specificity Study using Specific PCR
1539 Real-Time PCR Assay
40 RESULTS
1741 Bacterial Identification
1942 Sensitivity Studies of Pure Culture of V parahaemolyticus
421 Specific PCR Analysis Targeting tlh-gene bull 419 from Tilapia fish prior u~e for Spiking
Purpose - ~
20422 Vibrio parahaemolyticus Cell Count by Hemocytometer
423 Spiking and Dilution Samples for Standard 21 Curve
43 Specificity Study using Specific PCR 23
44 RT-PCR Assay for Detection of tlh positive 24 V parahaemolyticus in Cockles Samples
2650 DISCUSSION
60 CONCLUSION 30
REFERENCES 31
IV
35 APPENDICES
-
v
LIST OF TABLES
Table 1 PCR parameters
Table 2 The mixture was subjected to 35 cycles on a thermo cycler (Eppendorfreg Mastercycle Personal)
Table 3 Bacterial strains for specific PCR
Table 4 Cycling conditions ofRT-PCR of 40 cycles on Rotor Gene 6000 Corbett (Australia)
Table 5 RT-PCR reagent component
Table 6 Raw data for spiking samples with standard deviation value for standard curve
Table 7 Raw data for initial concentration for spiking samples
Table 8 Raw data for serial dilution samples with standard deviation value for standard curve
Page
11
12
15
15
16
35
35
36
VI
--------
LIST OF FIGURES
Page
Figure I The green colonies of V parahaemolyticus isolates on TCBS 18 agar plate
Figure 2 The mauve colonies of V parahaemolyticus isolates on 18 CHROMagartrade Vibrio
Figure 3 Agarose gel electrophoresis of the specific PCR pattern for 19 detection of Tilapia fish (Oreochromis niloticus) sample used prior to spiking using primers MV2B-tl which is tlhshybased gene targeting thennolabile hemolysin (tlh) gene Lanes L-l kbp DNA ladder (Invitrogen) I-Positive control (V parahemolyticus) 2-Tilapia fish sample 3-Non-template control
Figure 4 Hemocytometer count of undiluted V parahaemolyticus 20 culture viewed under 400x magnification
Figure 5 Standard curves for the number of V parahaemolyticus cells 21 versus Ct values of spiking samples The error bars indicate the standard deviations in two independent experiments
Figure 6 Standard curves for the number of V parahaemolyticus cells 22 versus Ct values of serial dilution samples The error bars indicate the standard deviations in two independent experiments
Figure 7 Agarose gel electrophoresis of the specific PCR pattern for 23 specificity test of MV2B-tl primer with other bactlrial strains Lanes L-l kbp DNA ladder (Invitrogen) I-Non-template control 2-Positive control (V parahaemolyticus) 3-Cockle sample 4-Fish sample 5-Escherichia coli (A TCC 56021) 6shyEscherichia coli KOll (ATCC 55124) 7-Saccharomyces cerevisiae (A TCC 24859) 8- Cryptococcus curvatus (ATCC 20509) 9- Gloephyllum trabeum (A TCC 11539) 10shyPhanerochaete chrysosporium (ATCC 24725) 11shySalmonella flexneri (ATCC 12022)
Figure 8 Melting curve analysis from 3 cockle samples with resulted 24 number of fluorescence against temperature
Figure 9 Quantification analysis from 3 cockle samples with resulted 25 number of cycle versus the number of fluorescence units for each sample used to determine Ct
VII
LIST OF ABBREVIATIONS
APW Alkaline Peptone Water
ATCC American Type Culture Collection
em centimetre
Ct Threshold cycle
DNA Deoxyribonucleic acid
dNTPs deoxynucleoside triphosphates
EDTA Ethylenediamine tetra-acetic acid
epsM extracellular secretion protein
EtBr Ethidium bromide
g gram
kbp kilo base pair
LB Luria Bertani
m meter
MgCh magnesium chloride
min minute(s)
mL mililiter
mM milimolar
NaCI sodium chloride
run nanometer
PCR Polymerase Chain Reaction
pH potential hydrogen
(shy correlation coefficient
rpm revoluhon per minute
VIII
RT-PCR
sec
TBE
TCBS
tdh
tlh
trh
UV
IJIll
~
middotC
Real-Time Polymerase Chain Reaction
second(s)
Tris-Borate-EDTA
Thiosulphate-Citrate-Bile Salts-Sucrose
thermostable direct hemolysin
thermolabile hemolysin
thermostable direct hemolysin related hemolysin
ultraviolet
micrometer
microliter
degree celsius
percentage
bull
IX
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
35 APPENDICES
-
v
LIST OF TABLES
Table 1 PCR parameters
Table 2 The mixture was subjected to 35 cycles on a thermo cycler (Eppendorfreg Mastercycle Personal)
Table 3 Bacterial strains for specific PCR
Table 4 Cycling conditions ofRT-PCR of 40 cycles on Rotor Gene 6000 Corbett (Australia)
Table 5 RT-PCR reagent component
Table 6 Raw data for spiking samples with standard deviation value for standard curve
Table 7 Raw data for initial concentration for spiking samples
Table 8 Raw data for serial dilution samples with standard deviation value for standard curve
Page
11
12
15
15
16
35
35
36
VI
--------
LIST OF FIGURES
Page
Figure I The green colonies of V parahaemolyticus isolates on TCBS 18 agar plate
Figure 2 The mauve colonies of V parahaemolyticus isolates on 18 CHROMagartrade Vibrio
Figure 3 Agarose gel electrophoresis of the specific PCR pattern for 19 detection of Tilapia fish (Oreochromis niloticus) sample used prior to spiking using primers MV2B-tl which is tlhshybased gene targeting thennolabile hemolysin (tlh) gene Lanes L-l kbp DNA ladder (Invitrogen) I-Positive control (V parahemolyticus) 2-Tilapia fish sample 3-Non-template control
Figure 4 Hemocytometer count of undiluted V parahaemolyticus 20 culture viewed under 400x magnification
Figure 5 Standard curves for the number of V parahaemolyticus cells 21 versus Ct values of spiking samples The error bars indicate the standard deviations in two independent experiments
Figure 6 Standard curves for the number of V parahaemolyticus cells 22 versus Ct values of serial dilution samples The error bars indicate the standard deviations in two independent experiments
Figure 7 Agarose gel electrophoresis of the specific PCR pattern for 23 specificity test of MV2B-tl primer with other bactlrial strains Lanes L-l kbp DNA ladder (Invitrogen) I-Non-template control 2-Positive control (V parahaemolyticus) 3-Cockle sample 4-Fish sample 5-Escherichia coli (A TCC 56021) 6shyEscherichia coli KOll (ATCC 55124) 7-Saccharomyces cerevisiae (A TCC 24859) 8- Cryptococcus curvatus (ATCC 20509) 9- Gloephyllum trabeum (A TCC 11539) 10shyPhanerochaete chrysosporium (ATCC 24725) 11shySalmonella flexneri (ATCC 12022)
Figure 8 Melting curve analysis from 3 cockle samples with resulted 24 number of fluorescence against temperature
Figure 9 Quantification analysis from 3 cockle samples with resulted 25 number of cycle versus the number of fluorescence units for each sample used to determine Ct
VII
LIST OF ABBREVIATIONS
APW Alkaline Peptone Water
ATCC American Type Culture Collection
em centimetre
Ct Threshold cycle
DNA Deoxyribonucleic acid
dNTPs deoxynucleoside triphosphates
EDTA Ethylenediamine tetra-acetic acid
epsM extracellular secretion protein
EtBr Ethidium bromide
g gram
kbp kilo base pair
LB Luria Bertani
m meter
MgCh magnesium chloride
min minute(s)
mL mililiter
mM milimolar
NaCI sodium chloride
run nanometer
PCR Polymerase Chain Reaction
pH potential hydrogen
(shy correlation coefficient
rpm revoluhon per minute
VIII
RT-PCR
sec
TBE
TCBS
tdh
tlh
trh
UV
IJIll
~
middotC
Real-Time Polymerase Chain Reaction
second(s)
Tris-Borate-EDTA
Thiosulphate-Citrate-Bile Salts-Sucrose
thermostable direct hemolysin
thermolabile hemolysin
thermostable direct hemolysin related hemolysin
ultraviolet
micrometer
microliter
degree celsius
percentage
bull
IX
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
LIST OF TABLES
Table 1 PCR parameters
Table 2 The mixture was subjected to 35 cycles on a thermo cycler (Eppendorfreg Mastercycle Personal)
Table 3 Bacterial strains for specific PCR
Table 4 Cycling conditions ofRT-PCR of 40 cycles on Rotor Gene 6000 Corbett (Australia)
Table 5 RT-PCR reagent component
Table 6 Raw data for spiking samples with standard deviation value for standard curve
Table 7 Raw data for initial concentration for spiking samples
Table 8 Raw data for serial dilution samples with standard deviation value for standard curve
Page
11
12
15
15
16
35
35
36
VI
--------
LIST OF FIGURES
Page
Figure I The green colonies of V parahaemolyticus isolates on TCBS 18 agar plate
Figure 2 The mauve colonies of V parahaemolyticus isolates on 18 CHROMagartrade Vibrio
Figure 3 Agarose gel electrophoresis of the specific PCR pattern for 19 detection of Tilapia fish (Oreochromis niloticus) sample used prior to spiking using primers MV2B-tl which is tlhshybased gene targeting thennolabile hemolysin (tlh) gene Lanes L-l kbp DNA ladder (Invitrogen) I-Positive control (V parahemolyticus) 2-Tilapia fish sample 3-Non-template control
Figure 4 Hemocytometer count of undiluted V parahaemolyticus 20 culture viewed under 400x magnification
Figure 5 Standard curves for the number of V parahaemolyticus cells 21 versus Ct values of spiking samples The error bars indicate the standard deviations in two independent experiments
Figure 6 Standard curves for the number of V parahaemolyticus cells 22 versus Ct values of serial dilution samples The error bars indicate the standard deviations in two independent experiments
Figure 7 Agarose gel electrophoresis of the specific PCR pattern for 23 specificity test of MV2B-tl primer with other bactlrial strains Lanes L-l kbp DNA ladder (Invitrogen) I-Non-template control 2-Positive control (V parahaemolyticus) 3-Cockle sample 4-Fish sample 5-Escherichia coli (A TCC 56021) 6shyEscherichia coli KOll (ATCC 55124) 7-Saccharomyces cerevisiae (A TCC 24859) 8- Cryptococcus curvatus (ATCC 20509) 9- Gloephyllum trabeum (A TCC 11539) 10shyPhanerochaete chrysosporium (ATCC 24725) 11shySalmonella flexneri (ATCC 12022)
Figure 8 Melting curve analysis from 3 cockle samples with resulted 24 number of fluorescence against temperature
Figure 9 Quantification analysis from 3 cockle samples with resulted 25 number of cycle versus the number of fluorescence units for each sample used to determine Ct
VII
LIST OF ABBREVIATIONS
APW Alkaline Peptone Water
ATCC American Type Culture Collection
em centimetre
Ct Threshold cycle
DNA Deoxyribonucleic acid
dNTPs deoxynucleoside triphosphates
EDTA Ethylenediamine tetra-acetic acid
epsM extracellular secretion protein
EtBr Ethidium bromide
g gram
kbp kilo base pair
LB Luria Bertani
m meter
MgCh magnesium chloride
min minute(s)
mL mililiter
mM milimolar
NaCI sodium chloride
run nanometer
PCR Polymerase Chain Reaction
pH potential hydrogen
(shy correlation coefficient
rpm revoluhon per minute
VIII
RT-PCR
sec
TBE
TCBS
tdh
tlh
trh
UV
IJIll
~
middotC
Real-Time Polymerase Chain Reaction
second(s)
Tris-Borate-EDTA
Thiosulphate-Citrate-Bile Salts-Sucrose
thermostable direct hemolysin
thermolabile hemolysin
thermostable direct hemolysin related hemolysin
ultraviolet
micrometer
microliter
degree celsius
percentage
bull
IX
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
--------
LIST OF FIGURES
Page
Figure I The green colonies of V parahaemolyticus isolates on TCBS 18 agar plate
Figure 2 The mauve colonies of V parahaemolyticus isolates on 18 CHROMagartrade Vibrio
Figure 3 Agarose gel electrophoresis of the specific PCR pattern for 19 detection of Tilapia fish (Oreochromis niloticus) sample used prior to spiking using primers MV2B-tl which is tlhshybased gene targeting thennolabile hemolysin (tlh) gene Lanes L-l kbp DNA ladder (Invitrogen) I-Positive control (V parahemolyticus) 2-Tilapia fish sample 3-Non-template control
Figure 4 Hemocytometer count of undiluted V parahaemolyticus 20 culture viewed under 400x magnification
Figure 5 Standard curves for the number of V parahaemolyticus cells 21 versus Ct values of spiking samples The error bars indicate the standard deviations in two independent experiments
Figure 6 Standard curves for the number of V parahaemolyticus cells 22 versus Ct values of serial dilution samples The error bars indicate the standard deviations in two independent experiments
Figure 7 Agarose gel electrophoresis of the specific PCR pattern for 23 specificity test of MV2B-tl primer with other bactlrial strains Lanes L-l kbp DNA ladder (Invitrogen) I-Non-template control 2-Positive control (V parahaemolyticus) 3-Cockle sample 4-Fish sample 5-Escherichia coli (A TCC 56021) 6shyEscherichia coli KOll (ATCC 55124) 7-Saccharomyces cerevisiae (A TCC 24859) 8- Cryptococcus curvatus (ATCC 20509) 9- Gloephyllum trabeum (A TCC 11539) 10shyPhanerochaete chrysosporium (ATCC 24725) 11shySalmonella flexneri (ATCC 12022)
Figure 8 Melting curve analysis from 3 cockle samples with resulted 24 number of fluorescence against temperature
Figure 9 Quantification analysis from 3 cockle samples with resulted 25 number of cycle versus the number of fluorescence units for each sample used to determine Ct
VII
LIST OF ABBREVIATIONS
APW Alkaline Peptone Water
ATCC American Type Culture Collection
em centimetre
Ct Threshold cycle
DNA Deoxyribonucleic acid
dNTPs deoxynucleoside triphosphates
EDTA Ethylenediamine tetra-acetic acid
epsM extracellular secretion protein
EtBr Ethidium bromide
g gram
kbp kilo base pair
LB Luria Bertani
m meter
MgCh magnesium chloride
min minute(s)
mL mililiter
mM milimolar
NaCI sodium chloride
run nanometer
PCR Polymerase Chain Reaction
pH potential hydrogen
(shy correlation coefficient
rpm revoluhon per minute
VIII
RT-PCR
sec
TBE
TCBS
tdh
tlh
trh
UV
IJIll
~
middotC
Real-Time Polymerase Chain Reaction
second(s)
Tris-Borate-EDTA
Thiosulphate-Citrate-Bile Salts-Sucrose
thermostable direct hemolysin
thermolabile hemolysin
thermostable direct hemolysin related hemolysin
ultraviolet
micrometer
microliter
degree celsius
percentage
bull
IX
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
LIST OF ABBREVIATIONS
APW Alkaline Peptone Water
ATCC American Type Culture Collection
em centimetre
Ct Threshold cycle
DNA Deoxyribonucleic acid
dNTPs deoxynucleoside triphosphates
EDTA Ethylenediamine tetra-acetic acid
epsM extracellular secretion protein
EtBr Ethidium bromide
g gram
kbp kilo base pair
LB Luria Bertani
m meter
MgCh magnesium chloride
min minute(s)
mL mililiter
mM milimolar
NaCI sodium chloride
run nanometer
PCR Polymerase Chain Reaction
pH potential hydrogen
(shy correlation coefficient
rpm revoluhon per minute
VIII
RT-PCR
sec
TBE
TCBS
tdh
tlh
trh
UV
IJIll
~
middotC
Real-Time Polymerase Chain Reaction
second(s)
Tris-Borate-EDTA
Thiosulphate-Citrate-Bile Salts-Sucrose
thermostable direct hemolysin
thermolabile hemolysin
thermostable direct hemolysin related hemolysin
ultraviolet
micrometer
microliter
degree celsius
percentage
bull
IX
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
RT-PCR
sec
TBE
TCBS
tdh
tlh
trh
UV
IJIll
~
middotC
Real-Time Polymerase Chain Reaction
second(s)
Tris-Borate-EDTA
Thiosulphate-Citrate-Bile Salts-Sucrose
thermostable direct hemolysin
thermolabile hemolysin
thermostable direct hemolysin related hemolysin
ultraviolet
micrometer
microliter
degree celsius
percentage
bull
IX
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
Dlat pengiraan
Rapid detectioD and quantification of Vibrio parahaemolyticus in Cockles (Anadara granosa) by using Real-Time PCR
Patricia Rowena aDak Mark Baran Saging
Resource Biotechnology Program Faculty of Resource Science and Technology
University Malaysia Sarawak
ABSTRACT
Vibrio parahaemolyticus (V parahaemolyticus) is acknowledged as one of the most significant disease-causing foodbome pathogen that causes the gastroenteritis resulting in diarrhea from vulnerable patients The pathogenicity genes of V parahaemolyticus are thermostable direct hemolysin (tdh) and thermostable direct hemolysin related hemolysin (trh) However targeting a gene of thermolabile hemolysin (tlh) gene in this study is described in total V parahaemolyticus which may play its role in affecting the host and contribute to human health risks This research aims for V parahemolyticus in rapidly detecting and quantitatively determines the present and the level of the bacteria from 30 samples of cockles (Anadara granosa) purchased from Kuchingshy7~-Mile wet market by utilizing the Real-Time PCR In this study the primer MV2B-tl (tlh-gene based) and the SYBR Green PCR mastermix were used in Real-Time PCR assay and among all the 30 samples of cockles isolated there were no detectable quantification of V parahaemolyticus Quantification of V parahaemolyticus could be determined by the standard curve result obtained Spiking of Tiapia fish with Ixl07 CFUmL V parahaemolyticu aids in determining the sensitivity of the study by Real-Time PCR assay by standard curve plot obtained with ~ value of 099 which showed a good correlation Furthermore the specificity of the assay was confinned by testing with 7 non - Vibrio strains by specific PCR The SYBR Green PCR mastermix and primer set were developed as quantification tool of V parahaemolyticus to the traditional PCR method which needed post-peR result and is time consuming Therefore the development of Real-Time peR assay in analyzing the presence of bacteria in seafood is crucial to both seafood industries as well as to the consumers
Keywords Vibrio parahaemolyticus tlh-gene Anadara granosa peR Real-Time PCR
ABSTRAK
Vibrio parahaemolyticus (f parahaemolvticus) adalah dikenali sebagai salah satu patogen makanan yang penting dimana ia menyebabkan penyakit gastroenteritis berkaitan derllian kesannya seperti cirit-birit yang boleh dikesan daripada orang yang mudah terkena oleh kesan bakteri~ tersebut Gen-gen patogenik V parahqemolyticus adalah thermostable direct hemolysin (tdh) dan thermostable direct hemolysin related hemolysin (trh) Walaubagaimanapun kajian ini menekankan sasaran dalam mengesan gen thermolabile hemolysin (tlh) yang boleh ditemui di dalam kebanyakan V parahaemolyticus di samping ia juga memainkan peranan dalam memberi risiro kepada kesihatan man usia Kajian ini telah dijalankan untuk mengenalpasti dan juga mengira bilangan bakteria tersebut yang hadir dalam 30 sampel kerang (Anadara granosa) yang telah dibeli di pasar basah sekitar Kuching-Batu 7 dengan menggunakan teknik Real-Time PCR Primer MV2B-tl (lchas gen tlh) dan SYBR Green PCR mastermix yang telah digunakan bagi 30 sampel kerang menunjukkan hasil yang negatif bagi pengiraan bilangan f parahaemolyticus menggunakan kaedah Real-Time PCR Pengiraan bilangan f parahaemolyticus boleh dilihat daripada hasil standard curve Kepekaan kajian menggunakan Real-Time PCR boleh dilihat dengan mencampurkan lxJ07 CFUlmL V parahaemolyticus pada ikan 1ilapia dan hasilnya boleh diperolehi daripada plot standard curve dengan nilai hubung kait yang bagus iDiIU =099 Tambahan pula spesifikasi kajian ini telah dipastikan dengan menguji 7jenis strain bukan Vibrio dttngan menggunakan PCR yang specific Pengunaan SYBR Green PCR mastermix dan primer penting sebagai
E parahaemolyticus jika dibandingkan dengan penggunaan kaedah PCR biasa yang erlukan langkah seterusnya bagi hasil PCR tersebut dan memakan masa yang agak panjang Oleh itu pettggunaan lcaedah Real-Time PCR dalam analisa kehadiran bakteria di dalam makanan laut adalah sangat penting cepada kedua-dua induslri makanan laut dan juga pengguna
E parahaemolylicus gen-tlh Anadara granosa PCR Real-Time PCR
x
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
CHAPTER 1
Introduction
Seafood products of high protein content are a great significant contribution for
human nutritions globally However with the continual wave outbreaks of food-borne
disease related to seafoods consumption associated to pathogenic bacteria this raises
awareness towards the public health concern (Gubala and Proll 2006) The disease may lead
to fatality if appropriate treatment is not undertaken immediately (Huang et al 2009) Vibrio
parahaemolyticus (V parahaemolyticus) is indigenous to marine environments around the
world and is reported to be the causative organism of acute food-borne gastroenteritis as well
as septicemia in human by the consumption of raw or undercooked seafood and hence arise
public concerns (Ward and Bej 2006)
In this paper the aim of study is to determine the occurrence of etiological V
parahaemolyticU8 from seafood sample cockles which is one of the most popular seafood
preferred in local cooking ingredients by the human populations in Malaysia Samples were
collected randomly from different locations of wet markets in ~uching- i h Mile district Due
to increase in demand of seafood intake and the increase of marine surface temperatures that
may result to the occurrence and risk of Vibrio foodborne infections (Hassan et al 2012)
thus rapid detection and quantification of V parahaemolyticus can be determined by using
real-time peR
A major foodbome-disease caused by V parahaemolyticus is most common in Asia
and United States although there were only few cases stated in Europe Statistically food-
borne disease has ranked as the highest incidence case rate of 3617 per 100 000 populations
and of 001 per 100 000 populations that signifies the mortality rate in year 2009 (Malaysia
Health Fact 2009) Furthermore under a survey of total 80 people in Hat Yi city of Thailand
1
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
there were about one-third of the people indicated with diarrhea after cockles intake
(FAOIWHO2011)
The traditional diagnoses methods like the utilisation of Polymerase Chain Reaction
(PCR) which include the molecular and biochemical testing for isolation and identification of
organisms (Singh and Mohapatra 2008) is ultimately tedious laborious and costly to some
extend of study and hence it gives a less precise result (Huang et al 2009) In addition
DNA-based method Polymerase Chain Reaction (PCR) which uses the Agarose gel
electrophoresis is able to give end point detection but it is not very quantitative to the PCR
product and hence due to the currently developed fluorogenic chemistries a new method
acknowledged as real-time PCR (RT-PCR) has emerged for the rapid detection and
quantification of microorganisms like V parahaemolyticus Real-time PCR analysis provides
a rapid and sensitive detection of reaction products through the fluorescence emission
(Gubala and Proll 2006) Similarly a recent study also revealed that real-time based
quantitative peR strategies which monitor the amplification reaction are the most accurate
(Lyon 2001) Ward and Bej (2006) mentioned that Real-time PCR assay also offers rapid
detection of total and pathogenic V parahaemolyticus in seafqed thereby is essential to both
seafood industry and consumer by guaranteeing the seafood prior to its consumption Thus
the development of Real-time PCR acts as a promising tool for the rapid detection of bacteria
such as in this case for detecting V parahaemolyticus present in environment and food
samples
11Objectives
1 To rapidly detect the occurrence of V parahaemolyticus in cockles (Anadara granosa) by
1argeting the thermolabile hemolysin (tlh) gene using Real-Time (RT -PCR) method
To quantify V parahaemolyticus by using Real-Time (RT-PCR)
2
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
CHAPTER 2
LITERATURE REVIEW
21 Vibrio Genus
Vibrio can be characterized as a significant group in the family of Vibrionaceae This
genus is mainly found in the marine environment whereby some Vibrios are pathogenic to
human Basically Vibrios are short Gram-negative facultative anaerobes that are motile and
possess single polar flagellum and having curve rod shaped of 14 - 18 11m in its average
length (Kelly et ai 1991 Ambrose 2005)
Generally Vibrios can be divided into two groups by characterisation non-halophilic
and halophilic According to Kass and Riemann (2006) Nishibuchi and DePaola (2005)
Vibrio parahaemolyticus and Vibrio cholerae are the Vibrio species that cause severe
intestinal disease Vibrios do tolerate in alkaline pH but is sensitive towards acid pH They
show their best growth in both pH and temperature ranging from pH 50 - 96 and 30 - 43degC
with the optimum rate of pH 76 and 37degC respectively (lCMSF 1996) ~
There are so far 12 species have been reported to be pathogenic to humans in which
among these species V parahaemolyticus and V cholerae and V vulnificus are those
associated with foodbome infections of the gastrointestinal tract (FAO 2011)
Vibrio parahaemolyticus is a Gram-negative non-spore forming bacterium
iOUHa1ive anaerobe that is able to grow in both pH and temperature ranging from pH 48 shy
_ 5 - 43degC with optimum pH 78 - 86 and 37 degC respectively (Lawley et al 2008)
3
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
lIDt~re
This organism possesses a single polar flagellum and is halophilic that grows best in I - 8
salt concentration in media (Sakazaki et al 2006) with optimum 3 (Lawley et al 2008)
There are two distinct groups of V parahaemolyticus namely Kanagawa-positive strains
which is pathogenic and causes food poisoning and Kanagawa negative strains which is
known to be hannless (Lawley et al 2008) Besides Ward and Bej (2006) mentioned that
there is the emergence of pandemic V parahaemolyticus associated with gastroenteritis
which is the 03K6 serotype It has also been reported that 03K6 serotype has caused a
number of outbreaks in many different states of the world (Nair et al 2007) V
parahaemo~yticus strains will normally produce green non-sucrose-fermenting colonies on
solid mediwn such as Thiosulphate-Citrate-Bile Salt-Sucrose (TCBS) agar (Sakazaki et aI
2006)
V parahaemolyticus is most profoundly be found in both estuarine and temperate
coastal marine environment globally The organism is a free living or may act as commensals
to other living aquatic organisms like shellfish and fish (Ward and Bej 2006) A correlation
to the latter statement fact also is discussed according to Sakazaki et al (2006) that V
parahaemolyticus adheres to chitin through chitinase secrelio~ thus has the ability to lay on
surface of shellfish and zoo-plankton Apart from that V parahaemolyticus favors warmer
temperature and is often related to seafoods from estuarine and coastal marine environment
8Dd therefore will show a greater risk of food poisoning linked to V parahaemolyticus
Cockles or also known as kerang in Malay is a marine bivalve mollusc which is a
aquatic product in Malaysia It can be classified into the genus Anadara from
family hence Anadara granosa (A granosa) is the scientific name and this species
4
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
Pusat Khidmlt Mlklumat Akademik UNlVERSm MALAYSIA SARAWAK
are mainly widespread in the warm temperate areas In addition A granosa is able to adapt
living in environment of relatively low salinity of 14 - 30 with 20 - 30 middotC of its optimum
temperature in 20 m water depth This intertidal species usually live shallowly buried in
sands and silts bottom Typically A granosa is measured of 3 cm length and can reach 5 - 6
em which in adult stage when harvested and they normally feed on organic detritus
phytoplankton and unicellular algae at the bed bottom of where they live (FAO 2011)
14 Route of Transmission of V parahaemolyticus
The wide distribution of V parahaemolyticus in nature and in manne coastal
environment especially precludes the possibility from reservoir to infection According to
Potasman et al (2002) the intake of raw molluscan shellfish which concentrate on bacteria
from environment through filter-feeding process leads to human infections Another
important issue of the transmission route is understood that this bacterium is generally
transmitted through the consumption of raw improperly cooked or cooked recontaminated
fish and shellfish particularly oysters Less commonly this 0l~anism can cause an infection
in the skin when an open wound is exposed to warm seawater (Daniels et al 2000)
COnical Manifestation of V parahaemolyticus
V parahaemolyticus causes food-borne infection known as gastroenteritis (Daniels et
2000) This type of gastroenteritis infection in human may be observed as mild to
iDoCltrate and is a self-limiting infection and the fatality rate is very low (Ray and Bhunia
In some cases of an affected human the onset of gastroenteritis symptoms that follows
48 hours upon the ingestion of V parahaemoiyticus include hyper secretion
5
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
gastroenteritis nausea vomiting chills fever watery diarrhea as well as abdominal cramps
(Yeung and Boor 2004) Moreover a report done by FDA (2010) claimed that once the
bacteria are ingested the incubation period required for symptoms to be apparent is from 4 shy
96 hours The scenario of V parahaemolyticus to cause gastroenteritis is by colonising in the
small intestine of the host and excrete toxin However in more severe cases there is a need
for a treatment either by fluid replacement or using antibiotics in order to reduce the recovery
period (park et al 2004)
26 Virulence properties of V parahaemolyticus
The involvement of pathogenicity factors like thermostable direct hemolysin (tdh) and
thennostable direct hemolysin related hemolysin (trh) are responsible for the infection by V
parahaemolyticus to be successful (Ray and Bhunia 2008) However it was also reported
that thennolabile hemolysin gene (tlh) may also be responsible for total V parahaemolyticus
aeleC110n since it is presence in all V parahaemolyticus strains although the gene is
(IOIDeI1I0W not related with pathogenicity (Ward and Bej 2006) bull-
Apart from that there were cases involving V parahaemolyticus in the outbreaks of
I118troentleritis related to seafood consumptions and this occur in various states include Spain
__n Japan Russia India North America and Southeast Asia (Chiou et al 2000
iMPlllniSl et al 2000 Lozano-Leon et al 2003 Okuda et al 1997 Smolikova et al 2001
1999 Yamazaki et aI 2003) Furthermore under a survey of total 80 people in Hat
of Thailand there were about one-third of the people indicated with diarrhea after
6
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
IecIlmlclue that
27 Isolation and Identification
Selective enrichment with Luria Bertani broth (LB broth) is recommended to enhance
the isolation of bacteria as the enrichment media works to increase recovery of the bacteria
from environmental stress and other samples (Wong 2002) Besides that alkaline peptone
water (APW) has been used as enrichment medium for the bacteria (Sakazaki et al 2006)
Since V parahaemolyticus is a halophilic organism (Sakazaki et al 2006) therefore it is
recommended that optimum 3 salt concentration (Curtis 2006) is added into the
enrichment media On top of that both TCBS agar and CHROMagartrade Vibrio are two
regularly used media for V parahaemolyticus isolation V parahaemolyticus strains will
normally produce green non-sucrose-fermenting colonies on TCBS medium (Sakazaki et al
2006) while on CHROMagartrade Vibrio medium the formation of mauve colonies will
indicate the presence of V parahaemolyticus The detection of colonies morphologies grown
on CHROMagartrade Vibrio is superior to the classical use ofTCBS agar (Kudo et al 2001)
28 Real-Time Polymerase Chain Reaction (RT-PCR)
Real-Time Polymerase Chain Reaction (R T -PCR) IS a rapid specific sensitive
somewhat evaluate the bacterial presence by quantitative means The
IMlDtifiCllltiClln of pathogenic microbes and their actions are crucial in accordance with their
detection (Dorak 2006) Hence RT-PCR is the continuous accumulation of fluorescent
amplification of peR product throughout cycles thus decreasing analytical time and
In addition fluorogenic probes target-gene-specific sequences internal to the primer
thus RT-PCR provide enhanced specificity as compared to conventional PCR-based
(Blackstone et al 2003) In Real-Time PCR the commonly used reaction
include the peR amplification reaction buffer magnesium chloride
7
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
deoxynucleoside triphosphate (dNTPs) DNA primers DNA polymerase and fluorogenic
probe Basically a complete amplification cycle involves denaturation annealing and
extension steps for the template DNA to be replicated (Dorak 2006) The first step involve
denaturation step which is crucial in separating the double stranded of DNA double helix in a
temperature between 94 - 95degC for 10 seconds Next is the annealing step that occurs at 60
middotC for 12 seconds to ensure the combined DNA primers with their complementary sequences
in target DNA Lastly the cycle is completed with the extension step at 72degC for 30 seconds
in which the polymerase extends from the primer in 5 to 3 direction The accumulated
fluorescence in reaction tube will be measured and usually this involve about 40 - 50 cycles
(Blackstone et al 2003)
Moreover there were researches which have been conducted by many researchers by
applying the Real-Time peR technique in detecting and quantifying V parahaemolyticus
One of the research conducted by Ward and Bej (2006) they had detecting V
ptuahemolyticus in shellfish particularly in oysters in United State by utilising Multiplexed
with TaqMan fluorescent probes Besides that in Malaysia Vengadesh et al (2012)
analysis for -deecting epsM gene from V
JlJllrJIRJMP in seafood
8
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
31 Sample coUection
In this study
monthly (3 months)
CHAPTER 3
MATERIALS AND METHODS
a total of 30 samples of cockles (Anadara granosa) were collected
randomly from different wet market in Kuching-i h Mile area Locations of sampling sites
include Stutong wet market and 7th Mile wet market All of these samples were collected
from the month of December 2011 until February 2012 During
JI1Dpling all samples taken were kept in polysterene box containing ice which act as a
temporary storage for cockles to maintain its freshness until analysed immediately in the
SlIIIple preparation and enrichment of bacterial sample
Cockle samples of25 g meat and fluid were prepared for homogenize process using a _bel bag for 60 seconds Prior to this cockle samples were transferred into 225 mL -
lbliine peptone water (APW) (1 peptone 1 NaCl) (MERCK Germany) so as to
ilia the growth of V parahaemolyticus As for the enrichment of bacterial samples
liIIIIii~ve and negative control) they were perfonned by using LB broth (Millers LB broth
laboratories) in order to enhance their growth Five hundred microliter of each
samples tested was inoculated into a universal bottle containing 10 mL LB broth
_Mirl8l bottles containing bacterial species were incubated in an incubator-shaker
BrumlWi(~ Innova model 4000) for overnight at 37 DC
9
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
33 Genomic DNA preparation
The protocol for isolation of total genomic DNA by boiling method is as described by
Noorlis et al (2011) Initially about 1 mL homogenate was aliquoted into a 15 mL
microcentrifuge tube and centrifuged (EBA 21 Hettich Zentrifugen) at 800 rpm for 1 minute
in order to separate the meat particle Next the supernatant was obtained and was subjected
to 12000 rpm for 3 minutes centrifugation to pellet the bacterial cells Supernatant was then
removed and 500 ~ sterile distilled water was added into the microcentrifuge tube
containing pellet The content was mixed by vortexing the tube gently The microcentrifuge
tube was then introduced to boiling at 100 DC for 10 minutes Subsequently the tube was
immerged in ice for 10 minutes at -20 DC and following that the tube was centrifuged again
at 13 000 rpm for 3 minutes Finally the supernatant was transferred into a new
microcentrifuge tube to be used as the DNA template and was kept at -20 DC until further use
bull Real-Time PCR assay The number of DNA template obtained for each cockles sample
- tive isolation and detection of Vibrio parahaemolyticus
The overnight enriched bacterial sample (V parahaemolyticus) cultured in LB broth
LB broth CONDA laboratories) was then streaked onto TCBS (MERCK
1liftII~It agar using a sterilised inoculating loop The streaked plates were incubated
__ at 37degC in incubator (SHELLAB incubator) The growth and the morphology of
i__riIl culture were observed and recorded Further confirmation of the selected single
the bacterial culture in which V parahaemolyticus produced green colonies on
IPu was then streaked onto CHROMagartrade Vibrio (Paris France) using a sterilized
10
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
inoculating loop and incubated overnight at 37degC Again the result was observed and
teCOrded
oS Polymerase Chain Reaction (PCR)
peR was performed to detect the specificity of the primer known as MV2B-tl primer
bich is tlh gene-based of the targeted virulence gene of V parahaemolyticus used in this
study and also tested for specificity with other bacterial strains as listed in Table 3 The PCR
~_nmque used is based on Zulkifli et al (2009) with slight modifications About 3 ilL of
extracted DNA sample were added into a 25 ~ PCR reaction mixture 40 ~ of lOx
buffer 30 ~ of 50 mM MgCh (Invitrogen) 10 JL of 10 mM 200 JL dNTPs mix
BioScience Taiwan) 30 ~ of V parahaemolyticus forward and reverse primers 10
of 5 Il cent Taq DNA Polymerase (Recombinant) (Ferment as Canada) The PCR
IIIIJtlifitcation was performed on a thermocycler (poundppendorfreg Mastercyc1e Personal) which
lIW~lveld pre-denaturation step at 96degC for 5 minutes 35 cycles of amplification which
IU_ denaturation step at 94degC for 1 minute annealing at63 degc and extension at 72degC
1minute 30 seconds and lastly final extension step at 72 degc for 7 minutes
Table 1 peR parameters
Volume per reaction 40
iDUUClcosiltie Triphosphate (dNTPs) 10 _ Chloride (MgCh) 30
ftriI_ (Forward) 30 1I1miml~ (Reverse) 30
10 30 110 250
11
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12
Table 2 The mixture was subjected to 35 cycles on a thermocycler (Eppendorfreg Mastercycle Personal)
Steps T emperatureTime Pre-denaturation 96degC (5 Min) Denaturation 94 degC (1 Min) Annealing 63degC (1 Min 30 Sec) 35 cycles Extension 72 degC (I Min 30 Sec) Final Extension 72degC (7 Min)
6 Agarose Gel Electrophoresis
Amplified DNA was subjected to electrophoresis process in which a 13 agarose gel
io-Rad Laboratories Spain) was prepared with Ix Tris-Borate-EDTA (TBE) buffer
(Promega Corp USA) PCR products of 5 ~ mixed with loading dye were loaded into each
with standard 1 kbp DNA ladder (Invitrogen) was used as a marker which loaded into
first well Reference strain of V parahaemolyticus obtained in laboratory was used as
illMitRre control Electrophoresis was carried out at a constant voltage of 110 Volt for an hour
an hour the gel was stained in Ethidium bromide (EtBr) for 15 minutes and destained
eel water for 10 minutes The amplified DNA fragments were then viewed under
1IIrIlyeni()1et (UV) transilluminator and result was analysed
fIIiM1ty study with pure culture for standard curve
1 middot6Ia aad bacterial cell count
bull ptUahaemolyticus known to be presented by growing culture on both TeBS and
Vibrio (Paris France) was subjected to grow in 250 mL LB broth (Millers
_~IIII1lOa model 4000) incubator-shaker at 37degC for 24 hours Prior to dilution step
12