ORIGINAL PAPER

A multiplex PCR assay based on 16S rRNA and hly for rapiddetection of L. monocytogenes in Milk

Ashwani Kumar • Sunita Grover • Virender Kumar Batish

Received: 2 October 2013 / Accepted: 23 March 2014

� Springer Science+Business Media New York 2014

Abstract A multiplex PCR assay was developed by tar-

geting ‘16S rRNA’ and ‘hly’ genes for detection of Listeria

or Listeria monocytogenes in dairy foods on the basis of

amplification of 1200 and 713 bp products, respectively.

The assay conditions were optimized to make it truly rapid

and to cut down the cost. The authenticity of the multiplex

PCR was ascertained by using Nested PCR targeted against

internal region of ‘hly’ gene that produced an amplified

product of 188 bp. The multiplex PCR assay was found to

be specific for detection of L. monocytogenes only since

none of the non-listerial cultures gave positive signal. The

sensitivity of the multiplex PCR was limited to 10 ng pure

DNA and 1–10 cells of L. monocytogenes after 4–6 h

enrichment in Listeria enrichment broth. When applied to

20 raw milk and 10 pasteurized milk samples, L. mono-

cytogenes could not be detected in any of the samples by

the multiplex PCR assay. This assay could find potential

application in dairy industry for monitoring dairy foods for

this high risk food pathogen on routine basis.

Keywords Multiplex PCR � 16S rRNA � hly �L. monocytogenes � Detection � Milk

Introduction

Listeria monocytogenes has emerged as a high risk food

pathogen of considerable public health significance on

account of its involvement in several outbreaks of listeri-

osis [6, 9] as well as gastrointestinal illnesses with the

consumption of contaminated processed and ready to eat

foods. It accounts for *1,700 cases of human illness and

650 deaths annually [7]. Being a facultative intracellular

pathogen, it is able to replicate inside the infected host cells

causing meningitis accompanied by septicemia, encepha-

litis, urethritis, gastroenteritis, endocarditis etc. [12] in

special risk groups of pregnant women, newborne, elderly

and immuno-compromised individuals. It has also pre-

sented a significant challenge to the processed food

industry in the recent years because of its ability to grow

over a wide range of temperature, pH and survive high

saline conditions [18]. Processed and ready to eat foods

such as milk, soft cheese, cooked poultary products, sea

foods etc. have been particularly implicated in outbreaks

and among these foods, consumption of dairy products has

been recognised as an important transmission route for

human listeriosis [1, 13]. Hence, timely and accurate

detection of this high risk food pathogen in foods partic-

ularly highly perishable dairy products is extremely

important from public health point of view.

Since the recognition of pathogenic potentials of L.

monocytogenes, a number of methods came into existence

for its isolation in clinical, food and environmental sam-

ples. A plethora of enrichment broths and selective agar

media are available, the selectivity of which is based on

specific phenotypic characteristics, namely, aesculin

hydrolysis, hemolytic activity, phosphotidyl-insitol specific

phospholipase C activity etc. [23, 24]. Later, immunolog-

ically based assays were also evolved for detection of

L. monocytogenes based on flagellar and surface antigenic

determinants. The use of immunomagnetic separation

(IMS) to concentrate Listeria from enrichment broths prior

to detection using Listeria TEK ELISA and a latex

agglutination assay targeting Listeriolysin ‘O’ monoclonal

antibodies (HDSE 1207; IgG2b) covalently bound to

A. Kumar � S. Grover � V. K. Batish (&)

Molecular Biology Unit, Dairy Microbiology Division, National

Dairy Research Institute, Karnal 132001, Haryana, India

e-mail: vkbatish@gmail.com

123

Food Measure

DOI 10.1007/s11694-014-9176-5

polystyrene amidine modified latex were developed for

detection of L. monocytogenes in foods [2, 3]. However,

these assays suffered from being laborious and the proba-

bility of identifying a specific pathogen is very low among

large background of nontargeted bacterial population [14].

PCR has completely taken over the aforesaid techniques

and have revolutionized the field of diagnostics with its

most outstanding applications for the detection of food-

borne pathogens. A number of genes including those

associated with virulence factors responsible for patho-

genesis exhibited by L. monocytogenes have been targeted

for PCR [4, 8, 15, 16, 20] and PCR based assays have been

developed for detection of L. monocytogenes with varying

degree of success when applied on foods. In this study, we

made an attempt to combine two sets of known primers

targeted against 16S rRNA (Listeria) and ‘hly’ (L. mono-

cytogenes) genes to develop a multiplex PCR for rapid

detection of L. monocytogenes in dairy foods.

Materials and methods

Bacterial cultures and maintenance

The bacterial cultures used in this investigation included

pathogenic strains of L. monocytogenes along with some

non-listerial cultures. Listeria monocytogenes ATCC 7644

was purchased from oxoid culture media and L. monocyt-

ogenes Scott A was procured from DM Division, NDRI,

Karnal. All the other cultures, viz., Salmonella typhi, Shi-

gella dysentriae, Shigella flexneri, S. aureus, E. coli

O157:H7, Lactobacillus delbrueckii subsp. bulgaricus,

Lactococcus lactis subsp. lactis were procured from

National Collection of Dairy Cultures, NCDC, Karnal,

India. The above mentioned cultures except lactic acid

bacteria were propagated in BHI (Brain Heart Infusion)

broth at 37 �C for 18 h. The cultures were preserved on

BHI agar slants and stored in refrigerator or as glycerol

stocks stored at -70 �C until further use. The cultures were

activated in BHI broth prior to use by sub-culturing at

biweekly intervals. The lactic cultures were preserved in

litmus milk tubes and activated in M17 and MRS broths

prior to their use.

Preparation of template DNA

Broth cultures

The template/genomic DNA was prepared from broth

cultures of L. monocytogenes by following boiled lysate

method [26] as well as the method of Pospeich and Neik-

mann [21]. The boiled lysate was prepared by harvesting

the overnight grown culture of the test organism followed

by heating the bacterial suspension in 50 ll MilliQ water

for 5 min in a boiling water bath and then centrifuging for

5 min at 10,000 rpm to separate the supernatant containing

DNA. For Pospeich and Neikmann’s method, the cells

were harvested from one ml of overnight grown cultures of

the test L. monocytogenes cultures and resuspended in

0.5 ml of SET buffer (75 mM NaCl, 25 mM EDTA,

20 mM Tris) and lysozyme was added at a concentration of

1 mg/ml (25 mM Tris, lysozyme, 10 mg, 5 M NaCl) fol-

lowed by incubation at 37 �C for 1 h. The subsequent step

was the addition of 1/10th volume of 10 % SDS and

0.5 mg/ml of proteinase K and incubation further contin-

ued for 2 h at 55 �C. One third volume of 5 M NaCl and

one volume of chloroform was added and incubated at

room temperature for 30 min with frequent inversions. The

samples were centrifuged and aqueous phase transferred to

a new tube and the DNA was precipitated by adding one

volume of isopropanol or two volumes of ethanol. The

DNA was pelleted, dried and dissolved in TER buffer

containing 10 lg/ml of RNase A. The sensitivity of the

PCR assay was determined by using different concentra-

tions (1000–1 ng) of L. monocytogenes DNA as template.

For determining the sensitivity in terms of cells, BHI broth

was inoculated with L. monocytogenes Scott A at the level

of 106, 105, 104, 103, 102, 10 and 1 cfu/ml and template

DNA from each sample was extracted as described above.

Spiked skim milk samples

The Non-fat Dry Milk (NFDM) was reconstituted @ 11 %

by dissolving 11 g of milk powder (NFDM) in distilled

water, autoclaved at 121 �C for 15 min and dispensed in

aliquots of one ml each in sterilized eppendorf tubes. The

aliquots of sterilized skim milk were then separately

inoculated with L. monocytogenes Scott A at the level of

107, 106, 105, 104, 103, 102, 10 and 1 cfu/ml. The DNA

from spiked milk samples, before and after enrichment (4

and 6 h) in Listeria enrichment broth, was then extracted

by following the ‘NDRI method’ previously developed in

our laboratory [22]. In brief, the steps included solvent

treatment in order to remove the fat layer from the milk

samples. 1 ml aliquots from the samples were then cen-

trifuged and the pellet dissolved in 400 ll of GTC buffer

and 400 ll of phenol saturated with TE (pH 8.0). Chloro-

form extraction was then carried out and DNA pelleted as

described above.

Raw and pasteurized milk samples

The raw and pasteurized milk samples were collected

aseptically from the local market. The samples were first

pre-enriched for 6 h in Listeria enrichment broth. Template

DNA was extracted from 10 ml aliquots of milk samples

A. Kumar et al.

123

before and after enrichment in LEB using the above

described ‘NDRI’ method. The samples were also analysed

microbiologically using LEB agar for typical L. monocyt-

ogenes colonies.

PCR assay

The PCR amplification for detection of L. monocytogenes

was performed using Eppendorf Mastercycler gradient,

5331, Germany. The selected oligonucleotide primers for

detection of L. monocytogenes were got custom synthe-

sized (Bangalore Genei, India). The description of the

primer pairs namely Lm3 and Lm5 targeting 16S rRNA

gene [25], ELMHLYFand ELMHLYR targeting ‘hly’ i.e.

hemolysin gene and ILMHLYF and ILMHLYR [15] tar-

geting internal region of hemolysin gene used in this study

is given in Table 1. The PCR assay was performed in 25 ll

reaction mixture comprising of 100 ng of template DNA,

109 PCR buffer (containing MgCl2), 0.2 mM (each of

primers), 0.2 mM dNTPs and 1 unit of Taq polymerase

(Boehringer Mannheim). Appropriate positive and negative

controls with each reaction were also set up. The PCR

parameters included initial denaturation at 95 �C for 4 min

followed by 35 cycles of denaturation at 94 �C for 30 s,

annealing at 60 �C for 1 min and extension at 72 �C for

1 min and final extension at 72 �C for 5 min.

Optimization of annealing temperature for multiplex PCR

amplification

Amplification conditions were optimized with respect to

annealing temperature (Gradient PCR using 60 �C with a

gradient of 2 �C).

Nested PCR

An internal set of primers ILMHLYF and ILMHLYR tar-

geted against hemolysin gene [15] was explored in order to

develop a nested PCR. A gradient PCR was set up with

annealing temperature ranging from 58 to 62 �C for 30 s to

confirm the authenticity of multiplex PCR amplified

product. The nested PCR assay was performed in 25 ll

reaction mixture comprising of 0.5 ll of 1:10 diluted PCR

amplified product from multiplex PCR as template, 109

PCR buffer (containing MgCl2), 0.2 mM (each of primers),

0.2 mM dNTPs and 1 unit of Taq polymerase (Boehringer

Mannheim). The PCR amplification conditions included

initial denaturation at 95 �C/4 min followed by 35 cycles

each of denaturation at 94 �C for 30 s, annealing at 60 �C

for 30 s and extension at 72 �C for 1 min followed by an

additional extension at 72 �C for 10 min.

Specificity of multiplex PCR assay

The specificity of the multiplex PCR assay was tested against

L. monocytogenes Scott A, L. monocytogenes ATCC 7644

and non listerial cultures namely S. typhi, S. dysentriae,

Shigella boydii, S. flexneri, Yerisinia enterocolitica, E. coli

O157:H7 and Lactic cultures namely Lactococcus lactis ssp.

lactis, Lactococcus lactis ssp. lactis biovar diacetylactis,

Lactococcus lactis ssp. cremoris, Streptococcus thermophi-

lus, L. delbrueckii ssp. bulgaricus etc.

Sensitivity of multiplex PCR

The sensitivity of multiplex PCR assay was tested in terms

of concentrations of template DNA (L. monocytogenes)

and cell number as descirbed above.

Analysis of PCR products

The PCR amplified products were electrophoresed on 2 %

agarose gel containing 0.5 lg/ml of ethidium bromide. The

gel was visualised under UV transilluminator and photo-

graphed using Polaroid 667 packfilm with MP4 system

polaroid camera (Photodyne, USA). The molecular size

marker i.e. 100 bp DNA ladder was also run on the gel to

monitor the size of the amplified PCR products.

Results and discussion

Advances in nucleic acid based detection methods offer an

opportunity to develop highly sensitive and specific

Table 1 Description of primers used in the present investigation

S. no. Target gene Primers Primer sequence Size of amplified

product (bp)

References

1 16S rRNA Lm 3 50-ggA CCg ggg CTA ATA CCg AAT gAT AA-30 1200 Wiedmann et al. [25]

Lm 5 50-TTC ATg TAg gCg AgT TgC AgC CTA-30

2 Hemolysin ELMHLYF 50-TCC gCC TgC AAg TCC TAA gA-30 713 Klein and Juneja [15]

ELMHLYR 50-gCg CTT gCA ACT gCT CTT TA-30

ILMHLYF 50-gCA ATT TCg AgC CTA ACC TA-30 188 Klein and Juneja [15]

ILMHLYR 50-ACT gCg TTg TTA ACg TTT gA-30

Multiplex PCR for detection of L. monocytogenes

123

methods for the detection of foodborne pathogens includ-

ing L. monocytogenes quickly. In this context, PCR based

techniques have revolutionized the detection of food

pathogens in dairy foods like milk, ice-cream, soft cheeses

etc. Development of such PCR based techniques could be

an extremely valuable and practical approach for rapid

detection of L. monocytogenes in foods. These methods in

general offer an advantage for the detection of L. mono-

cytogenes in milk and other dairy foods because they can

detect the presence of even stressed or injured cells which

would be unrecoverable by conventional culture methods.

Such methods have potential application in dairy industry

as routine screening tools in quality assurance labs. In this

study, we also explored PCR technology for developing a

reliable, sensitive and specific assay for rapid detection of

L. monocytogenes in dairy foods by targeting 16S rRNA

gene and hemolysin gene. 16S rRNA gene has been tar-

geted in this study for the identification of all Listeria

species at genus level [11, 17]. The choice for targeting

16S rRNA gene has been dictated by the presence in

microorganisms of multiple copies (104) of rRNA, thereby,

increasing the ease of signal generation of the assays. The

16S rRNA based primers included in the multiplex PCR

resulted into the amplification of 1.2 kb product specific for

Listeria. The choice of second primer set for the multiplex

PCR assay in this investigation was based on the capability

of L. monocytogenes to produce hemolysin which is

involved in the lysis of vacuole and erythrocytes. The main

factor involved in the lysis of vacuole is the protein Lis-

teriolysin O, that has pore forming activity and is encoded

on ‘hly A’ gene. The primer pair namely ELMHLYF and

ELMHLYR targeted against ‘hly’ gene when used in the

PCR assay produced an amplified product of 713 bp with

template DNA from L. monocytogenes only.

Optimization of PCR amplification parameters

for multiplex PCR

Since rapidity of the multiplex PCR assay are going to be

very crucial factors for its practical utility, attempts were

made initially to optimize the PCR amplification conditions

and meticulous selection of other PCR parameters.

Annealing temperature

The first step in this direction was initiated by setting a

gradient PCR to find the most optimal annealing temper-

ature at which the recovery of the two amplified PCR

products i.e. 1200 and 713 bp was maximum. A gradient

PCR using annealing temperature of 60 �C with gradient of

2 �C was set up. The temperature in different tubes were

58.1, 58.2, 58.5, 58.9, 59.4, 59.9, 60.5, 61.0, 61.5, 61.9,

62.1 �C as the Eppendorf machine has already the software

for setting the temperature by giving gradient. The agarose

gel analysis of the PCR amplified samples clearly indicated

that 1200 and 713 bp products could be detected in all the

samples subjected to a gradient of annealing temperature

ranging from 58.1 to 62.1 �C. However, the best amplifi-

cation appears to be at 59.9 and 60.5 �C, both in terms of

intensity and sharpness of bands (Data not shown). Hence,

for further studies, 60 �C was selected as the best annealing

temperature for incorporation in the multiplex PCR. Our

studies clearly indicate that the two primers incorporated in

the multiplex PCR assay worked quite effectively at a wide

range of annealing temperature, thereby, indicating quite a

bit of flexibility in the annealing temperatures for further

manipulations as and when desired. Our results in this

regard are consistent with the observations of Klein and

Juneja [15] who also used an annealing of 60 �C in their

multiplex PCR using the same ‘hly’ gene specific primers

as used in our study. However, our results in relation to the

use of Lm3 and Lm5 set of primers targeted against 16S

rRNA in our multiplex PCR are at variance from those of

Wiedmann et al. [25] who found 55 �C as the optimal

annealing temperature in their PCR assay performed with

only one set of primers. This variation may not be signif-

icant since we consistently observed a reasonable amplifi-

cation of both the products at 60 �C in our multiplex PCR.

Since, the annealing temperature of 60 �C worked quite

well in our assay, no further efforts were made to reduce

the same to 55 �C as was used by Wiedmann et al. [25].

Authentication of multiplex PCR products by nested

PCR

In classical PCR assays, the identity of specific PCR pro-

ducts is generally checked by monitoring on agarose gel

and subsequent confirmation by southern hybridization

with a labeled probe highly specific for the targeted gene.

Sometimes the identity of the PCR product is further

ensured by digestion of the PCR products with restriction

endonucleases whose recognition sequence is supposed to

be with in the amplified product. Application of these steps

in conjunction with conventional PCR assays could be

counter productive as far as the rapidity of the PCR assay is

concerned by negating the advantages of this technology.

Hence, to make PCR based techniques a real time propo-

sition for rapid detection of food pathogens, there is a need

to find quick methods for ascertaining the authenticity of

the PCR based assays. Recently, there has been lot of

interest in using nested PCR for doing the needful.

Standardization of nested PCR

In our multiplex PCR, one of the L. monocytogenes specific

gene that was targeted was ‘hly’ and a PCR amplified

A. Kumar et al.

123

product of 713 bp specifically associated with L. mono-

cytogenes was obtained along with 1200 bp product

attributed to genus specific 16S rRNA gene. For ascer-

taining the L. monocytogenes origin of the 713 bp ampli-

fied PCR product in our multiplex PCR, we explored a set

of internal primers targeted within the region in place of

southern hybridization (which makes use of a labeled L.

monocytogenes specific probe) to cut short the lengthy time

required in such hybridization experiments. However, we

invariably detected additional bands along with the genuine

188 bp product on agarose gel when we set up PCR reac-

tion with such internal primers using 0.5 ll of the amplified

PCR product of our own multiplex PCR as the template as

such. Hence, to resolve this discrepancy, an attempt was

made to optimize the nested PCR reaction conditions by

varying the concentration of the template and the annealing

temperature as described below.

Dilution of template for nested PCR

Since template concentration could be a critical factor in

nested PCR, effect of dilution (1:10) of the template DNA

with different aliquots (0.5, 1, 2, 5 ll) of amplified product

obtained from multiplex PCR with L. monocytogenes

template DNA was determined on the amplification of

‘hly’ internal primer in the nested PCR. Our results clearly

indicated that the 188 bp product that represented the true

amplified product of 713 bp template derived from the

external hly primers, was produced exclusively only when

0.5 ll of the 1:10 diluted template DNA was used (Data

not shown). However, when the volume of the diluted

sample was raised to 1 ll or more or 0.5 ll of the undiluted

template was used in the reaction, the 188 bp product was

invariably associated with 713 bp band on the agarose gel,

thereby, suggesting that the 713 bp band had come directly

from the template as a carry over rather than amplification

since the intensity of this band was always weak. Never-

theless, to avoid any confusion and ambiguity in the

results, we choose 0.5 ll of the 1:10 diluted amplified

product of our multiplex PCR as the ideal template con-

centration for nested PCR to confirm unequivocally that the

713 bp product produced in our multiplex PCR assay was

indeed of L. monocytogenes origin.

Optimization of annealing temperature for nested PCR

In order to further optimize the amplification of 188 bp

product in the nested PCR, the effect of different annealing

temperatures was also studied by running a gradient nested

PCR using a wide range of annealing temperature (58–62 �C

for 30 s). The results pertaining to the same are illustrated in

Fig. 1. As can be evidenced from the agarose gel picture, the

188 bp product could be detected at all the annealing

temperatures used in the experiment (Fig. 1, lanes 1–11),

thereby, indicating greater flexibility in the selection of

annealing temperature in the nested PCR. However for our

convenience, we choose 60 �C for 30 s as the annealing

temperature/time combination for 25 cycles in the nested

PCR assay. Based on these results the optimal nested PCR

parameters were found to be initial denaturation at 95 �C for

4 min followed by 25 cycles each of denaturation at 95 �C

for 4 min, annealing at 60 �C for 30 s, extension at 72 �C for

30 s along with additional extension step at 72 �C for 5 min.

Our findings on the use of nested PCR can be compared with

those of Klein and Juneja [15] who explored nested PCR to

produce L. monocytogenes specific probes by targeting the

internal regions located in the ‘iap’ and ‘hly’ genes which

were also previously used for development of multiplex RT-

PCR assays for detection of viable L. monocytogenes. By

using these internal sets of primers, these investigators were

able to achieve the amplification of 119 and 188 bp products

which they finally used as probes for confirming the detec-

tion of viable L. monocytogenes. Our strategy used for nested

PCR was different from that of Klein and Juneja [15] in the

sense that we used the 713 bp PCR amplified product from

our multiplex PCR as the template instead of genomic DNA

from L. monocytogenes used directly by the later. From these

results, we were able to unequivocally prove the detection of

L. monocytogenes in spiked milk samples and pure broth

cultures. The development of such nested PCR-based assays

could prove to be an asset to dairy industry for confirming the

presence of L. monocytogenes in dairy foods quickly to leave

adequate time to take appropriate follow up action. This

would not only strengthen the reliability of PCR based assays

but also can go a long way in protecting the health of con-

sumers from L. monocytogenes.

Specificity of multiplex PCR assay

The specificity of the multiplex PCR assay was also

checked in this study to rule out the possibility of false

positive results. For this purpose, the genomic DNA

Fig. 1 Effect of annealing temperature on amplification of internal

‘Hly’ Primers with nested PCR for confirmation of L. monocytogenes.

Lanes M marker; 1–11: 1 58.1, 2 58.2, 3 58.5, 4 58.9,5 59.4, 6 59.9, 7

60.5, 8 61.0, 9 61.5, 10 61.9, 11 62.1, 12 positive control (60 �C), 13

negative control

Multiplex PCR for detection of L. monocytogenes

123

extracted from a large number of non listeria cultures was

used as a template in the multiplex PCR. The results per-

taining to the effect of template from the non targeted

organisms on the amplification of the L. monocytogenes

specific PCR products in the multiplex PCR assay have

been presented in Fig. 2. As is quite evident from the gel

picture, the two specific bands 713, 1200 bp supposed to be

unique for L. monocytogenes could not be detected with

any of the non targeted cultures which include E. coli,

Salmonella, Shigella, Yersinia, Campylobacter, Lactococci

and Lactobacilli etc. These results clearly point towards

high specificity of the assay, thereby, ruling out the pos-

sibility of any false signals attributed to nonspecific factors.

However, the specificity of the assay could not be assessed

against other Listeria spp. other than L. monocytogenes due

to non availability of those cultures. Our results concerning

the specificity are in accordance with those observed by

Klein and Juneja [15] who also found their RT-PCR tar-

geted ‘iap’, ‘hly’ and ‘prf’ highly specific for L. mono-

cytogenes only. However, these investigators had tested the

RT-PCR assay against different species of L. monocytog-

enes and did not include non Listerial cultures for evalu-

ating the specificity of the assay. We, on the other hand,

tested our multiplex PCR against a number of Gram

positive, Gram negative and non-Literial cultures as well,

none of which gave the positive signal with our multiplex

PCR assay. Our results with regard to specificity of 16S

rRNA based Listeria specific primers included in our

multiplex PCR can be further substantiated by almost

similar observations recorded by Wiedmann et al. [25] who

also found their PCR coupled LCR assay extremely spe-

cific for L. monocytogenes only.

Sensitivity of multiplex PCR assay for

L. monocytogenes detection

The sensitivity of multiplex PCR assay was determined at

three different levels with regard to nature of the template

i.e. with pure DNA of L. monocytogenes, DNA obtained

form BHI broth inoculated with L. monocytogenes at dif-

ferent levels (Boiled lysate) and DNA extracted from milk

samples spiked at different levels of L. monocytogenes by

‘NDRI’ method.

Sensitivity with pure DNA

Initially, we determined the sensitivity of the multiplex

PCR assay by using different concentrations (1–1,000 ng)

of the pure DNA preparation of L. monocytogenes. The

results regarding the same have been presented in Fig. 3.

From the agarose gel picture presented therein, it is quite

clear that the positive signal in terms of formation of both

the bands i.e. 1200 and 713 bp could be detected unam-

biguously even at as low DNA concentration as 10 ng

(Fig. 3, lanes 1–6). Even with 1 ng DNA concentration,

weak signal could be observed on the gel, although the

same could not be reproduced in the gel picture. Hence, we

presume our multiplex PCR works well at a minimal

concentration of 10 ng, when pure preparation of DNA was

used as the template in the assay. The results pertaining to

the sensitivity of our multiplex PCR assay when different

levels of pure DNA were used as template cannot be

substantiated due to the non availability of any possible

report on these lines. But nevertheless, this limitation does

not underestimate the significance of our findings. Based

on these results, we can safely recommend that our mul-

tiplex PCR assay is sensitive enough to pickup signals even

with as low as 10 ng of pure DNA from L. monocytogenes

thus making it of considerable practical significance.

Sensitivity in terms of L. monocytogenes cells in broth

culture

The sensitivity of the multiplex PCR assay was also deter-

mined in terms of detection of cells of L. monocytogenes in

Fig. 2 Specificity of multiplex PCR against targeted cultures other

than Listeria monocytogences. Lanes M 100 bp Marker, 1, E. coli

0157:H7; 2, E. coli K-12; 3, S. flexneri; 4, Shigella boydii; 5, S. typhi;

6, S. dysentriae; 7, S. aureus; 8, B. cereus; 9, Campylobacter jejuni;

10, Y. enterocolitica; 11, Lactococcus lactis subsp. lactis; 12, L.

delbrueckii subsp. bulgaricus; 13, L. monocytogenes; 14, negative

controlFig. 3 Sensitivity of multiplex PCR assay for detection of L.

monocytogenes in terms of DNA concentration. Lanes M Marker,

1–7 (Template DNA, ng/ll): 1 1000, 2 5000, 3 250, 4 100, 5 50, 6 10,

7 1, 8 negative control

A. Kumar et al.

123

BHI broth inoculated at different levels both before and

after 4 and 6 h enrichment in Listeria enrichment broth at

37 �C. The results regarding the same have been presented

in Fig. 4. It can be inferred from the gel picture that both

1200 and 713 bp bands could be detected when boiled

lysates from 105, 104, 103, 102 cells (without enrichment)

were used as template for the multiplex PCR assay (Fig. 4,

lanes 1–4). However, the positive signal was not detected

with 10 cells in the broth (Fig. 4, lane 5), thereby, indi-

cating that the sensitivity of our assay with pure broth

culture was limited to 100 cells only. However, after 4 h

enrichment in LEB, the multiplex PCR assay could detect

as low as 10 cells as revealed by the formation of 1200 and

713 bp bands on the gel (Fig. 4, lane 8). The sensitivity

could be further enhanced to 1 cell after 6 h enrichment

(Fig. 4, lane 12). Our results in this context appear to be

almost comparable to those of Deneer and Boychuk [10]

who could detect as low as 5–50 cfu of L. monocytogenes

by a PCR assay involving two rounds of 35 amplification

cycles without need for subsequent hybridization with

labeled probes. Contrary to this, Klein and Juneja [15] were

able to detect different levels of L. monocytogenes by RT-

PCR carried out with different sets of primers targeted

against iap, hly, prf genes. Among these, iap based RT-

PCR assay was the most sensitive as it could detect as low

as approximately 10–15 cfu from pure culture after 1 h

enrichment. However, detection of 713 hly specific

amplicon was approximately less sensitive after 1 h

enrichment, where as detection of prf A product showed

the lowest level of sensitivity.

Sensitivity in terms of cell number in spiked milk

samples

The main objective of this study was to develop a multiplex

PCR assay for eventual application in dairy foods for rapid

detection of L. monocytogenes. However, before applying

this assay on naturally contaminated milk samples, there is

a need to validate the working of the assay in dairy foods

artificially inoculated with L. monocytogenes. Hence, an

attempt was first made to test the sensitivity of this assay in

milk samples after spiking the foods artificially with the

targeted pathogen. In this context, initially we spiked

sterilized skim milk samples at different levels of L.

monocytogenes and prepared templates from these samples

before and after enrichment in LEB broth for 4–6 h by

following ‘NDRI’ extraction protocol. The results of mul-

tiplex PCR reaction set up with the templates obtained

from spiked milk samples before and after enrichment have

been presented in Fig. 5. As it is quite evident from the gel

picture, both 1200 and 713 bp products specific for Listeria

and L. monocytogenes could be clearly detected with

samples spiked at the level of 107,106,105,104 cells without

enrichment (Fig. 5, lanes 1–4). However, the same cate-

gory of samples spiked at 103 cells failed to show the two

bands (Fig. 5, lane 5), thereby, indicating the sensitivity of

our multiplex PCR limited to 104 cells/ml. Contrary to this,

introduction of 4–6 h enrichment steps prior to PCR

resulted into dramatic increase in the sensitivity of multi-

plex PCR assay since as low as 100 cells could be cleanly

detected after 4 h enrichment which was further reduced to

even 1–10 cells after 6 h enrichment as has been indicated

in Fig. 5 (lanes 6–8) for 4 h samples and (lanes 10–12)

after 6 h enrichment. Our results in this regard appear to be

consistent with observations of Cooray et al. [8] who were

able to detect as low as 0.1 cfu of L. monocytogenes in

milk by their PCR assay after 24 h preenrichment in LEB

and Listeria plating media. The exceedingly high sensi-

tivity of these investigators can be ascribed to inordinately

long enrichment which may be counterproductive to the

rapidity of the PCR assay. Our results can be further sup-

ported from almost similar observations made by Cano

et al. [5] who could find lowest level of sensitivity of the

PCR-FD assay between 10 and 100 cfu. Nogva et al. [19]

on the other hand were able to get much higher level of

sensitivity (6 cfu/ml) with their 50-nuclease PCR assay

when used in conjunction with Dynal antilisterial beads in

skim milk and unpasteurised whole milk. In a related study,

following a different strategy involving integration of IMS

and polycarbonate membrane capture in their PCR assay

targeted against 16S-23S IGSR of L. monocytogenes,

O’Connor et al. [20] were able to detect as low as 1–10 cfu

per 25 ml in inoculated raw and pasteurised milk samples

after overnight enrichment.

From these results, it can be concluded that the sensi-

tivity of a particular PCR assay is specifically dependent on

the nature of the primers used in the assay which might be

the cause of variations in the sensitivities of different

multiplex PCR assays used in the detection of L. mono-

cytogenes. Moreover, the enrichment of samples for dif-

ferent intervals can influence the sensitivity of the assay

Fig. 4 Sensitivity of multiplex PCR assay for detection of L.

monocytogenes in BHI broth inoculated with different levels of

target cell. Lanes M 100 pb Marker: 1–5 (cfu/ml; Before enrichment):

1 10,0,000; 2 10,000; 3 1,000; 4 100; 5 10;6–9 (cfu/ml; 4 h

enrichment) 6 1,000; 7 100; 8 10; 9 1; 10–12 (cfu/ml; 6 h

enrichment): 10 100; 11 10; 12 1; 13 Positive control, Scott A; 14

negetive control

Multiplex PCR for detection of L. monocytogenes

123

significantly as can be observed from our results. Never-

theless, our results do demonstrate that the sensitivity of

our multiplex PCR was fairly high and can be considered

even better obtained with other studies since simply by

introducing a short (4–6 h) enrichment step before the PCR

reaction could clearly detect as low as 10 cells from spiked

milk samples, thereby, enhancing the practical applicability

of this assay.

Raw milk

In this study, a total of 20 raw milk samples were analysed

with multiplex PCR assay for detecting the possible pre-

sence of L. monocytogenes. To ensure the workability of

the assay, some of these samples were also spiked with L.

monocytogenes and included in this study as positive

controls. The results of multiplex PCR in respect of seven

of the selected raw milk samples before and after spiking

with targeted pathogen followed by 6 h enrichment in LEB

have been illustrated in Fig. 6. As is quite evident from the

agarose gel picture, none of the seven unspiked raw milk

samples (Fig. 6, lanes 1, 2, 3, 5, 7, 9 and 11) showed the

presence of Listeria or L. monocytogenes since neither

1200 bp nor 713 bp product could be detected on agarose

gel with these samples even after 6 h enrichment. How-

ever, when 5 of the samples were spiked with L. mono-

cytogenes, the two bands (Fig. 6, lanes 4, 6, 8, 10 and 12)

could be clearly detected on the gel, thereby, indicating

that the negative results obtained with raw milk samples

were genuine. In fact, all the 20 raw milk samples tested by

our multiplex PCR showed negative results and appear to

be free of L. monocytogenes. The absence of L. monocyt-

ogenes signals in these samples with the multiplex PCR

could also be confirmed by microbiological analysis of the

samples using LEB agar as the plating medium on which

no suspected Listeria colonies could be recovered with any

of the samples. Our results in this regard are contradictory

from those of Cano et al. [5] who were able to detect L.

monocytogenes from 25 out of 53 samples with their PCR-

FD assay and found a good correlation between PCR and

cultural results. The possible reasons for variations in the

two studies may include complete absence of L. mono-

cytogenes from the raw milk samples we had examined in

our study or their contamination at a very low level which

may not be detectable by our PCR assay or inadequate

recovery of the template from raw milk samples by ‘NDRI’

method. Although, the possibility of such negative reac-

tions obtained with raw milk samples in this study appears

to be quite remote, it can not be completely ruled out.

Perhaps an extended enrichment of these samples could

have provided answer to this problem but at the cost of

delayed results which we could not afford since the main

objective of study was to develop a rapid method for

prompt detection of L. monocytogenes in dairy foods.

Pasteurised milk

In this study we included ten pasteurized milk samples also

for application of a multiplex PCR assay for detecting the

possible presence of L. monocytogenes. Based on our

results, L. monocytogenes could not be detected from any

of the ten pasteurized milk samples (Data not shown) since

the two specific amplified product viz 1.2 kb and 713 bp

could not be detected with any of the samples indicating

that all the ten pasteurized samples were not contaminated

with L. monocytogenes. The results of our multiplex PCR

assay on pasteurized milk samples cannot be substantiated

due to non-availability of any published report on these

lines. The possible reasons for negative results with all the

pasteurized milk samples examined in this study by mul-

tiplex PCR assay may include absence of the pathogen

from such samples, exceedingly low level of L. monocyt-

ogenes cells unable to provide adequate amount of tem-

plate for PCR amplification or inefficient processing of

template from pasteurized milk samples which might

require some additional treatments to recover L. monocyt-

ogenes specific template in PCR amenable form.

Fig. 5 Sensitivity of multiplex of PCR assay for detection of

L. monocytogenes in milk inoculated with different levels of target

cells. Lanes M 100 bp Marker; 1–5 (cfu/ml, before enrichment); 1

107; 2 106; 3 105; 4 104; 5 103; 6–9 (cfu/ml, 4 h enrichment): 6 103; 7

102; 8 10; 9 1, 10–12 (cfu/ml, 6 h enrichment): 10 103; 11 102; 12 10;

13 1; 14 Negative control

Fig. 6 Appliction of multiplex PCR assay for monitoring raw milk

samples for L. monocytogenes. Lanes M 100 bp Marker; 1 RM-1; 2

RM-2, 3 RM-3, 4 RM-3 (spiked), 5 RM-4, 6 RM-4 (spiked), 7 RM-5,

8 RM-5 (spiked), 9 RM-6, 10 RM-6 (spiked), 11 RM-7, 12 RM-7

(spiked), 13 Lm Scott A, 14 negative control

A. Kumar et al.

123

From the foregoing study, it can be concluded that our

multiplex PCR assay based on 16SrRNA and hly genes is

rapid, reliable and sensitive enough to detect as low as

1–10 cells of L. monocytogenes in dairy foods in less than

12 h. Hence, this assay could find potential application in

dairy industry for monitoring dairy foods for this high risk

food-pathogen on routine basis.

Acknowledgments The authors duly acknowledge the support

received from The Director, NDRI, Karnal and DBT, Govt. of India

for carrying out this work. The technical assistance provided by Mr.

Inder Kumar, Technical Officer is also acknowledged.

References

1. L.R. Beuchat, Pathogenic microorganisms associated with fresh

produce. J. Food Prot. 59, 204–216 (1996)

2. R.R. Beumer, M.C. Tegiffel, M.T.C. Kok, L.J. Cox, F.M.

Rombouts, A comparison of rapid methods for the detection of

Listeria monocytogenes. Food Aust. 48, 160–162 (1996)

3. N. Bruchez, J.L. Cordier, Detection of Listeria monocytogenes

using immunomagnetic beads (vicam-Lister test), in Proceeding

of the XII International Symposium on Problems of Listeriosis.

Promaco Conventions Pty. Ltd., Canning Bridge, Western Aus-

tralia, pp 251–252

4. A. Bubert, I. Hein, M. Rauch, A. Lehner, B. Yoon, W. Goebel,

M. Wagner, Detection and Differentiation of Listeria spp. by a

single reaction based on multiplex PCR. Appl. Environ. Micro-

biol. 65, 4688–4692 (1999)

5. R.J. Cano, D.M. Norton, A.E. Inzunza, J.G. Sanchez, C. Oste,

Polymerase chain reaction assay coupled with fluorescence

detection on microwell plates for L. monocytogenes in foods.

J. Food Prot. 58, 614–620 (1995)

6. CDC, Food borne outbreak of Listeriosis associated with pork

tongue jelly. Morb. Mortal. Weekly Rep. 49, 221–222 (2000)

7. J. Cooper, R.D. Walker, Listeriosis. Vet. Clin. North Am. Food

Anim. Prac. 14, 113 (1998)

8. K.J. Cooray, T. Nishibori, H. Xiong, T.C. Matsuyama, M. Fujita,

M. Mitsuyama, Detection of multiple virulence-associated genes

of Listeria monocytogenes by PCR in artificially contaminated

milk samples. Appl. Enviorn. Microbiol. 60, 3023–3026 (1994)

9. C.B. Dalton, C.C. Austin, J. Sobel, An outbreak of gastroenteritis

and fever due to Listeria monocytogenes in milk. N. Engl. J. Med.

336, 100–105 (1997)

10. H.G. Deneer, I. Boychuk, Species-specific detection of Listeria

monocytogenes by DNA amplification. Appl. Environ. Microbiol.

57, 606–609 (1991)

11. J.M. Gopo, R. Melis, E. Filipska, J. Filipski, Development of a

specific biotinylated DNA probe for rapid identification of Sal-

monella. Mol. Cell. Probes 2, 271–280 (1988)

12. M.L. Gray, A.W. Killinger, Listeria Monocytogenes and Listeria

infections. Bacteriol. Rev. 30, 309–382 (1966)

13. P.S. Hayes, J.C. Feeley, L.M. Graves, G.W. Ajello, D.W.

Fleming, Isolation of Listeria monocytogenes from raw milk.

Appl. Environ. Microbiol. 51, 438–440 (1986)

14. A. Hurst, Bacterial injury: a review. Can. J. Microbiol. 23,

935–944 (1977)

15. P.G. Klein, V.K. Juneja, Sensitive detection of viable Listeria

monocytogenes by reverse transcription-PCR. Appl. Environ.

Microbiol. 63, 4441–4448 (1997)

16. A. Kumar, S. Grover, V.K. Batish, Monitoring paneer for Listeria

monocytogenes: a high risk food pathogens by multiplex PCR.

Afr. J. Biotechnol. 11(39), 9452–9456 (2012)

17. P. Maureau, I. Derclaye, D. Gregorie, M. Janssen, G.R. Cornelis,

Campylobacter species identified on polymorphism of DNA

encoding rRNA. J. Clin. Microbiol. 27, 1514–1517 (1989)

18. G. Mitchell, Y. Varabioff., L. monocytogenes,Y. enterocolitica:

their roles in food-borne disease. Dairy Prod. 8–13 (1988)

19. H.K. Nogva, K. Rudi, K. Naterstad, A. Holck, L. Dag, Applica-

tion of 50-nuclease PCR for quantitative detection of Listeria

monocytogenes in pure cultures, water, skim milk, and unpas-

teurized whole milk. Appl. Environ. Microbiol. 66, 4266–4271

(2000)

20. L. O’Connor, J. Joy, M. Kane, M. Maher, Rapid polymerase

chain reaction/DNA probe membrane based assay for the detec-

tion of Listeria and L. monocytogenes in food. J. Food Prot. 63,

337–342 (2000)

21. A. Pospiech, B. Neikmann, A versatile quick preparation of genomic

DNA from Gram-positive bacteria. TIG 11, 217–218 (1995)

22. S. Suresh Kumar, Detection of E. coli O157:H7 in milk by PCR.

Thesis submitted to National Dairy Research Institute (Deemed

University), NDRI, Karnal. (1999)

23. P. Van Netten, I. Perales, A. Van de Moosdijk, G.D.W. Curtis,

D.A.A. Mossel, Liquid and solid selective differential media for

the detection and enumeration of Listeria monocytogenes and

other Listeria species. Int. J. Food Microbiol. 8, 299–316 (1989)

24. G. Vlaemynck, V. Lafarge, S. Scotter, Improvement of the

detection of L. monocytogenes by the application of ALOA, a

diagnostic, chromogenic isolation medium. J. Appl. Microbiol.

88, 430–441 (2000)

25. M. Wiedmann, F. Barany, C.A. Batt, Detection of Listeria

monocytogenes with a nonisotopic polymerase chain reaction

assay. Appl. Environ. Microbiol. 59, 2743–2745 (1993)

26. P.K. Witham, C.T. Yamashiro, K.J. Livak, C.A. Batt, A PCR

based assay for detection of Escherichia coli shiga like toxin

genes in ground beef. Appl. Environ. Microbiol. 62, 53–1347

(1996)

Multiplex PCR for detection of L. monocytogenes

123