Journal of Microbiological Methods 98 (2014) 99–104
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Journal of Microbiological Methods
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Application of the VPp1 bacteriophage combinedwith a coupled enzymesystem in the rapid detection of Vibrio parahaemolyticus
Yong Peng a,1, Yanqiu Jin a,1, Hong Lin a, Jingxue Wang a,⁎, Muhammad Naseem Khan a,b
a Food Safety Laboratory, College of Food Science and Engineering, Ocean University of China, Qingdao 266003, Chinab Microbiological Analytical Centre, FMRRC, PCSIR Labs. Complex Karachi, 75280, Pakistan
For rapid and quantitative detection ofVibrio parahaemolyticus, amethod combining the specific lysis of bacterio-phages with a bacterial luciferase–flavin mononucleotide:nicotinamide adenine dinucleotide oxidoreductasebioluminescent system in vitro was developed. A V. parahaemolyticus detection systemwas established by opti-mizing three main influencing factors: bacteriophage titer, volume ratio of the bacteriophage to its host bacteri-um, and lysis time. A standard curve between the number of bacteria and the luminescence intensity of thecoupled enzyme system was studied and revealed a good linear relationship. More than 107 colony-formingunits (cfu)·ml−1 bacteria in pure culture and N108 cfu·ml−1 bacteria in oyster samples were readily detectedwithout pre-enrichment. Furthermore, N100 cfu·ml−1 bacteria in oyster samples were readily detected after4 h of enrichment culture. Because of its rapid detection, high specificity, and simplicity in operation, thismethodis an effective tool for detecting living bacteria in food and environmental samples.
The significant public health problem of foodborne infectious dis-ease has been constant or increasing in many countries during the lastdecade (Martinez-Urtaza et al., 2010). Previously, pathogenic bacteriain food were usually detected using traditional isolation and identifica-tionmethodswith drawbacks such as long detection period, impracticalprocedures, and poor specificity (Kaper et al., 1980; Sakazaki et al.,1979). In recent years, newmethods such as polymerase chain reaction(Pollini et al., 1997), enzyme-linked immunosorbent assay (Caruso etal., 2002), immunofluorescence (Chen and Chang, 1996), and gene-chip technology (He et al., 2005) have been developed to detect micro-organisms. These techniques have the advantages of sensitivity, rapidi-ty, and precision. However, some of them cannot be widely appliedowing to high cost or complexity. Nonspecific detection is a particularproblem in the detection of certain viable cells (Pinto et al., 2008).
Bacteriophages have been introduced in the development of thesemethods for the specific detection of pathogenic bacteria (Blasco et al.,1998). Phages are the natural enemies of bacteria and well known fortheir extreme host specificity, making them ideal candidates for appli-cations designed to detect target pathogenic bacteria (Hagens andLoessner, 2007). The virulent bacteriophage VPp1 was isolated fromaquatic sewage water (Peng et al., 2013), and the specific lysis of this
ononucleotide;NADH, nicotin-L, yellow light.
ghts reserved.
phage was combined with a bacterial luciferase–flavin mononucleo-tide:nicotinamide adenine dinucleotide (FMN:NADH) oxidoreductasebioluminescent system in vitro (also called the coupled enzyme systemestablished by the Food Safety Laboratory of Ocean University of China)for the rapid and quantitative detection of Vibrio parahaemolyticus.
Briefly, NADH released by the phage-specific lysis of host bacteriawas mixed with the coupled enzyme system and visible light at490 nm was emitted. In particular, bacterial luciferase and NADH:FMNoxidoreductase are important enzymes in luminous bacteria biolumi-nescent systems in vivo, and they catalyze an oxidation to emit lightat 490 nm in the presence of FMN, NADH, a long-chain aliphatic alde-hyde, and molecular oxygen (Hastings and Gibson, 1963; Hastings andNealson, 1977; Nealson and Hastings, 1979). These reactions can be de-scribed concisely as follows (Esimbekova et al., 2007; Karatani andKonaka, 1998):
FMNþNADHþ Hþ →FMN:NADH oxidoreductase
FMNH2 þNADþ ð1Þ
RCHOþ FMNH2 þ O2 →Bacterial luciferase
FMNþ RCOOHþH2Oþ hv: ð2Þ
Thenumber of viable cells is proportional to the luminescence inten-sity of the coupled enzyme system. Therefore, the number of targetpathogenic bacteria is obtained by converting the luminescence intensi-ty of the system. In this method, NADHwas used as the indicator owingto its pervasiveness and stability in all viable cells (Olek et al., 2002).NADH is also an essential substrate for the coupled enzyme system(Mei et al., 2009). Put simply, we studied a rapid, sensitive,
100 Y. Peng et al. / Journal of Microbiological Methods 98 (2014) 99–104
uncomplicated, and inexpensive method that combined specific bacte-riophages with the pervasiveness of NADH.
2. Materials and methods
2.1. Bacterial strains and culture media
A V. parahaemolyticus strain VP 17802 (ATCC 17802)was used as theprimary host for the isolation and propagation of bacteriophages(Fujino et al., 1974). A luminous yellow light (YL) bacterium(Photobacterium leiognathi) was isolated offshore in Qingdao, China, bythe Food Safety Laboratory of Ocean University of China. The strainwas deposited in the China Center for Type Culture Collection with thecollection number M 206139. The GenBank accession number isEF017227.
Other eleven strains of V. parahaemolyticus stored in our lab, VPVIB304, VP VIB461, VP VIB800, VP1.1, VP1.2, VP2.1, VP3.2, VP4.1, F3-3,M1, and M2, were used to detect the host range of bacteriophage(Table 1).
2216E liquid and 2216E agar (Hope Bio-Technology Co., Ltd., China)are the commonmedium formarine bacteria with themajor compoundas 0.5% tryptone, 0.1% yeast extract, 3.4% NaCl and 0.01% FePO4, pH 7.6–7.8.
2.2. Phage preparation
Aquatic sewage water samples were obtained from an aquatic prod-uct market in Qingdao. The samples were centrifuged at 5000 r·min−1
for 10 min, and 5 ml of the supernatant and 100 μl of early log-phasehost culture were added to 5 ml double-strength 2216E liquid mediumand incubated at 37 °C overnightwith shaking. Themediumwas centri-fuged, and the supernatant was filtered through a syringe filter (0.22-μm pore size; EMD Millipore Co., Billerica, MA, USA). The filtrate wasthen appropriately diluted with sterile SM buffer (5.8 g/l NaCl, 2 g/lMgSO4·7H2O, and 50 ml/l 1 M Tris–HCl, pH 7.5). The double-layermethod was used to test for the presence of phages (Adams, 1959;Terzaghi and Sandine, 1975).
2.3. Optimization and establishment of the coupled enzyme system
The luminescence intensity of P. leiognathi YL cells and the coupledenzyme system were measured with an Ultra-Weak Luminescence An-alyzer (BPCL_K, Institute of Biophysics, Chinese Academy of Sciences,Beijing, China) according to absorption at 474 nm and a time intervalof 1 s. The unit of the luminescence intensity was expressed in counts.The coupled enzyme system was established and optimized as 300 μlof sample liquid, 1 ml of crude enzyme, 0.5 μl of 10 mmol·l−1 FMN-Na, 100 μl of 27 mmol·l−1 dodecane, and 300 μl of 0.14 mmol·l−1
NADH (Mei et al., 2009).
Table 1Bacteria and host range.
V. parahaemolyticusstrains
Source/reference VPp1 infection
VP 17802 American Type Culture Collection +VP VIB304 LMG 2850T +VP VIB461 Shrimp +VP VIB800 Shrimp −VP1.1 Isolated from seawater −VP1.2 Isolated from seawater −VP2.1 Isolated from seawater −VP3.2 Isolated from seawater −VP4.1 Isolated from seawater −F3-3 Isolated from seawater −M1 Isolated from Ostrea plicatula −M2 Isolated from Ostrea plicatula −
+, VPp1 infected strain−, No infection.
2.3.1. Preparation of crude enzymeA single colony of P. leiognathi YL was inoculated in the 2216E liquid
medium (300 ml) and incubated at 25 ± 0.5 °C with a shaking at140 r·min−1. The medium was centrifuged at 4000 r·min−1 for15 min when luminescence intensity reached more than 3.0 × 106
counts, and then resuspended in 30 ml of phosphate-buffered saline(0.01 mol/l, pH 7.0, with 10.0 mmol/l EDTA, 1.0 mmol/l DTT). Resus-pension was followed by supersonic treatment of frequency 20 kHzfor 90 min with a disrupter (JY92-II, SIENIZ, Ningbo, China) in an icebath. Cellular debris was removed via centrifugation at 9000 r·min−1
and 4 °C for 30 min, and the supernatant was precipitated by the addi-tion of solid ammoniumsulfatewith the saturation degree between 40%and 80%. The precipitate was resuspended in 1 ml of Tris–HCl(0.02 mol/l, pH7.0). The preparation was then desalted with a chro-matographic column filled with Sephadex G-25 medium.
2.3.2. Quantitative detection of NADH with the coupled enzyme systemNADH standard liquid of various concentrations (300 μl) was added
to the coupled enzyme system (1 ml of crude enzyme with 0.5 μl of10 mmol·l−1 FMN-Na, 100 μl of 27 mmol·l−1 and dodecane). Lumi-nescence intensity was measured with the Ultra-Weak LuminescenceAnalyzer (BPCL_K) at 471 nm, and the time interval was 1 s. The unitof the luminescence intensity was expressed in counts. A blank controlexperiment was performed by adding 300 μl of phosphate-buffered sa-line to the coupled enzymes. Each assaywas performed three times. Thestandard curve was established between different concentrations ofNADH and the luminescence intensity of the coupled enzyme system.
2.4. Optimization of the lysis conditions of the bacteriophage
2.4.1. Bacteriophage titerA mixture of 1 ml of various bacteriophage titers (7.4 × 1010,
7.4 × 109, 7.4 × 108, 7.4 × 107, and 7.4 × 106 plaque-forming units[pfu]·ml−1) and 1 ml of early log-phase VP 17802 suspension wasmixed and incubated at 37 °C for 45 min with shaking. The lumines-cence intensity was detected with the coupled enzyme system using ablank control experiment. Each assay was performed three times.
2.4.2. Lysis timeBacterial cells in the stationary phase were collected via centrifuga-
tion at 5000 r·min−1 for 10 min, resuspended in sterile normal saline,and diluted to various concentrations (optical density at 600 nm valuesof 0.4, 0.8, and 1.2). One milliliter of bacteriophages (1010 pfu·ml−1)and 1 ml of VP 17802 suspension of various concentrations weremixed and incubated at 37 °C for 10, 15, 30, and 45 min.
2.4.3. Volume ratio between the bacteriophage and its host bacteriaExperimentswere carried out with various volume ratios of the bac-
teriophage and host cells (1:1, 1:3, 1:5, 3:1, and 5:1 respectively). Themixtures of bacteriophage (7.4 × 1010 pfu·ml−1) and VP 17802 sus-pension with various volume ratios were incubated at 37 °C for15 min with shaking. The luminescence intensity was detected withthe coupled enzyme system using a blank control experiment.
2.5. Establishment of a standard curve for the detection of V.parahaemolyticus with the detection system
Bacterial cells were collected and diluted into various densitygradients and detected with the coupled enzyme system combinedwith bacteriophage lysis. The number of cells was counted using aplate colony-counting method with a blank control experiment. Eachassay was performed three times. The standard curve was thenestablished between the number of VP 17802 cells and the lumines-cence intensity of the coupled enzyme system.
Fig. 1. Effects of bacteriophage titer on the detection system. Luminescence intensityvalues of different titer of phages are depicted in red, negative control values are shownin white. Average luminescence intensity and standard deviation (error bars) were de-rived from three features (n = 3) per assay.
101Y. Peng et al. / Journal of Microbiological Methods 98 (2014) 99–104
2.6. Sensitivity of the detection system for V. parahaemolyticus
The mixture of bacteriophage (7.4 × 1010 pfu·ml−1) and VP 17802suspensionwith a volume ratio of 1:1was incubated at 37 °C for 15 minwith shaking. The luminescence intensity was detected with thecoupled enzyme system, and the assay was performed 20 times. Themeans x and standard deviation (SD) of the results were calculated,and the lowest detected luminescence intensity was x + 3 s. The min-imumdetection number ofV. parahaemolyticuswas then obtained usingthe equation of linear regression of the standard curve in linear range.
2.7. Recovery experiments to detect V. parahaemolyticus in oyster sampleswith the detection system
2.7.1. Recovery experiments in pure cultureVP 17802 suspensions of various concentration were prepared with
bacterial content of approximately 0.31 × 108–5.03 × 108 colony-forming units (cfu)·ml−1 and then detected with the coupled enzymesystem combined with bacteriophage lysis. The recovery rate of eachgroup and average percentage of recovery were calculated.
2.7.2. Recovery experiments in oyster samplesOysterswere processed via the entry–exit inspection and quarantine
method of the People's Republic of China industry standard (SN/T 0173-2010) as follows. Oyster meat (25.0 g) was obtained using aseptic tech-nique and added to 225.0 ml of 2216E liquid medium. The mixture washomogenized in a blender (Joyoung, China) by blending for 30 s. Then,various concentrations of VP 17802 suspension were added and blend-ed, and the mixture was allowed to stand for 2 min. Supernatant(3.0 ml)was obtained andmixedwith 3.0 ml of bacteriophage in steriletubes. These tubes were incubated at 37 ± 0.5 °C with shaking at140 r·min−1 for 15 min. Then, 300 μl of lysis solution was used to de-tect the luminescence intensity with the coupled enzyme system. Ablank control experiment was performed using oyster samples withoutVP 17802. Each assay was performed in five parallel tests. A thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Luqiao Technology Co. Ltd.,China) platewas used to count the amount of VP 17802 in every sample.
2.8. Repeatability of experiments to detect V. parahaemolyticuswith the de-tection system
Tests to detect V. parahaemolyticus in pure culture suspension andoyster sampleswere repeated seven times each. The results were trans-lated into corresponding V. parahaemolyticus counts using the equationof linear regression of the standard curve. The relative SD of the resultswas calculated to analyze the repeated experiments.
2.9. Application of the detection system in seafood
2.9.1. Sample pretreatmentOystermeat (2.0 g)was obtained using aseptic technique and added
to 18 ml of 2216E liquidmedium. Themixture was homogenized. Then,various concentrations of VP 17802 suspension were added so that theconcentration of VP 17802 in the samples reached 1.6 × 108, 2.1 × 108,2.6 × 108, 3.2 × 108, 5.7 × 108, and 7.3 × 108 cfu·ml−1, respectively.The mixture was left stationary for 1 min, and then 3.0 ml of superna-tant was obtained and mixed with 3.0 ml of bacteriophage in steriletubes. These tubes were incubated at 37 ± 0.5 °C with shaking at140 r·min−1 for 15 min. Luminescence intensity was then measured.
2.9.2. Pre-enrichment experiment with V. parahaemolyticusVP 17802 in the stationary phase was collected and suspended at
various concentrations and added to 25 g of oyster meat obtainedwith aseptic technique with 225 ml of 2216E liquid medium. The con-centration of VP 17802 in every sample was 100 cfu·ml−1. Incubationfollowed at 37 °C with shaking and hourly sampling to determine
luminescence intensity using the coupled enzyme system combinedwith bacteriophage lysis. Thiosulfate-citrate-bile salts-sucrose agarplates were used to determine the amount of VP 17802 in every sample.The number of V. parahaemolyticus was computed using luminescenceintensity on the standard curve.
3. Results
3.1. Phage preparation
The lytic bacteriophage VPp1, previously isolated from sewagewater samples by our laboratory, was proved effective in lysing V.parahaemolyticus at a low multiplicity of infection (0.0001) with atiter of 1010 plaque-forming units (pfu)/ml, which has a double-strand-ed DNA, an icosahedral head of 44 nm in diameter, and no tail (Peng etal., 2013). The phage VPp1 belongs to the Tectiviridae family accordingto the International Committee on Taxonomy of Viruses (Fauquet et al.,2005). The strain was deposited in the China Center for Type CultureCollection with the collection number M 2013030.
A host range experiment was performed. Three of twelve strains ofV. parahaemolyticus could be lysed by bacteriophage VPp1 (Table 1).
3.2. Quantitative detection of NADH with the coupled enzyme system
The coupled enzyme system was highly sensitive to low concentra-tions of NADH. Prominent increase in luminescence intensity of thecoupled enzymes was observed by adding 0.1 nmol·l−1 of NADH. Agood linear relationship between NADH concentration and lumines-cence intensity was observed within the range of 0.1 to 12.0 nmol·l−1,and a standard curve was obtained (y = 237.21x + 3039.6) with apositive correlation coefficient (R2) of 0.9812 (Supplementary Fig. 1).The detection limit reached 0.1 nmol·l−1, which was similar to theresults published by Mei et al. (2009).
3.3. Optimization of lysis conditions of bacteriophages
3.3.1. Bacteriophage titerResults showed that as the phage titer increased, the luminescence
intensity also increased (Fig. 1). Therefore, the titer of7.4 × 1010 pfu·ml−1 was selected for the detection system because itwas generally and easily obtained in our experiment.
3.3.2. Lysis timeResults revealed that various VP 17802 suspension concentrations
displayed no obvious rise in luminescence intensity until 10 min oflysis. However, the luminescence intensity ascended markedly whenlysis time reached 15 min, and no further increases were seen withlysis times longer than 15 min. This result indicates that the bacterial
102 Y. Peng et al. / Journal of Microbiological Methods 98 (2014) 99–104
cells were completely lysed after 15 min. Therefore, the lysis time of thedetection system was determined to be 15 min (Fig. 2).
3.3.3. Volume ratio between the bacteriophage and its host bacteriaResults indicated that in various concentrations of VP 17802 suspen-
sion, luminescence intensity increased with increases in the volumeratio, reaching a maximum value and stabilizing when the volumeratio was 1:1 (Fig. 3). These results revealed that the bacterial cellswere completely lysed at volume ratio of 1:1. Therefore, the volumeratio of the bacteriophage and its host bacteria was determined to be1:1.
Fig. 3. Effects of the volume ratio between bacteriophage and host cells on the detectionsystem. Luminescence intensity values measured at 1:5 are depicted in red, 1:3 in yellow,1:1 in purple, 3:1 in green and 5:1 in blue. Negative control values are shown in white.Average luminescence intensity and standard deviation (error bars) were calculatedfrom three features (n = 3) per assay.
3.4. Standard curve of V. parahaemolyticus detection with the detectionsystem
Different bacterial counts of VP 17802 (3.1 × 107, 5.2 × 107,1.02 × 108, 1.91 × 108, 3.17 × 108, 4.25 × 108, 5.03 × 108, and6.18 × 108 cfu·ml−1) were detected by the detection system. As bacte-rial count increased, luminescence intensity increased stably. Moreover,a good linear relationship between bacterial count and luminescence in-tensity in the range of 3.1 × 107 to 6.18 × 108 cfu·ml−1 was observed.A standard curve was established between the total bacterial count ofVP 17802 and luminescence intensity. The standard curve showed a lin-ear relationship as described by y = 2030.4x + 2706.7 with an R2 of0.9838 (Fig. 4). Bacteria numbering above 107 cfu·ml−1 in pure culturewere readily detected without pre-enrichment. The entire detectionwas completed within 30 min, including bacteriophage lysis and lumi-nescence intensity detection.
3.5. Sensitivity of the detection system for V. parahaemolyticus
The mean of the results of 20 blank samples was 3033.4455, and theSDwas 46.7136. The lowest luminescence intensitywas 3173.5863, andthe minimum detection number of V. parahaemolyticus was2.30 × 107 cfu·ml−1.
3.6. Recovery experiments to detect V. parahaemolyticus in oyster with thedetection system
The average recovery rates of six experiments in pure culture andoyster samples were 96.28% (Supplementary Table 1) and 91.92% (Sup-plementary Table 2), respectively. The method showed high accuracy.
Fig. 2. Effects of the lysis time of bacteriophage on the detection system. Luminescence in-tensity valuesmeasured at 10 min are depicted in blue, 15 min in green, 30 min in yellow,and 45 min in red. Negative control values are shown in white. Average luminescence in-tensity and standard deviation (error bars) were calculated from three features (n = 3)per assay.
3.7. Repeatability of experiments to detect V. parahaemolyticuswith the de-tection system
The experimental results indicated that the relative SDs of V.parahaemolyticus in pure culture and oyster samples were 0.72% and0.68%, respectively. Both were less than 1%, which showed that themethod had high precision and good repeatability.
3.8. Application of the detection system in seafood
3.8.1. Sample pretreatmentThe entry–exit inspection and quarantinemethod of the People's Re-
public of China industry standard (SN/T 0173-2010)were implementedto detect V. parahaemolyticus in seafood. Twelve oysters were selectedand cleaned with running water. Ethanol (79%) was used to disinfectthe oyster shells and remove all meats (including viscera) and liquidwith an aseptic technique.
3.8.2. Pre-enrichment experiment with V. parahaemolyticusThe detection limits under the bacterial culture and oyster sample
conditions were 107 and 108 cfu·ml−1, respectively. However, the con-centration of V. parahaemolyticus in shellfish can be considerably low(Fuenzalida et al., 2006; Garcia et al., 2009). Thus, a pre-enrichment ex-periment was performed. As shown in Fig. 5, luminescence intensitywas unnoticeable for the first 3 h because VP 17802 was in a lagphase and displayed slow growth. Over time, the amount of bacteriagrew. Luminescence intensity was obvious after 4 h. A plate colony
Fig. 4. Standard curve of detecting the total bacterial count of Vibrio parahaemolyticus bythe coupled enzyme system and bacteriophage lysis. Dots represent the experimentaldata and solid line, the linear fit. Average luminescence intensity and standard deviation(error bars) were calculated from three features (n = 3) per assay.
Fig. 5. Determination of pre-enrichment time of Vibrio parahaemolyticus. Average lumines-cence intensity and standard deviation (error bars) were calculated from three features(n = 3) per assay.
103Y. Peng et al. / Journal of Microbiological Methods 98 (2014) 99–104
counting method showed that the number of bacteria was1.7 × 108 cfu·ml−1 at 4 h. In otherwords, an oyster samplewith an ini-tial concentration of 100 cfu·ml−1 can be detected by the coupled en-zyme system after 4 h of enrichment.
4. Discussion
The V. parahaemolyticus detection system used in this study wasbased on NADH as the indicator. Before detection, it is important tomaximize NADH extraction to determine bacterial quantity. To increasethe rate of fluorescence intensity and NADH stability, we chose a hotTris–HCl method according to Wang et al. (2009). Nevertheless, allmethods for obtaining NADH cannot accomplish the specific detectionof a target pathogenic bacterium—a challenge resolved by the introduc-tion of bacteriophages in this research. Bacteriophages are well knownfor their extreme specificity, and they can be used for biocontrol of bac-teria without interfering with natural microflora (Hagens and Loessner,2007).
This report is the first on the detection of V. parahaemolyticus usingbacteriophage lysis combined with a bacterial luciferase system. Thissystem has many advantages, such as rapidity, high specificity, and op-erational ease. In this study, the detection limit of NADH reference ma-terial was 10−10 mol·l−1 because the luciferase and FMN:NADHoxidoreductase were crude and lacked purification. Stanley (1971) hasreported that the detection limit of NADH reference material canreach 10−12 mol·l−1 when commercialized luciferase and FMN:NADHoxidoreductase are used. Consequently, further purification of luciferaseand FMN:NADH oxidoreductase will effectively increase sensitivity anddecrease detection limit. Additionally, an ATP bioluminescence methodhas already been thoroughly researched, and a kit and other portabledetection instruments used extensively in microorganism detectionhave been developed (Tanaka et al., 1997). NADH has a biolumines-cence reaction mechanism similar to that of ATP, which implies thatthe NADH bioluminescencemethodmay also be used to develop detec-tion kits, portable detection instruments, or other biosensors based onsystematic research of the further purification, co-immobilization,recycling, and stability of luciferase and FMN:NADH oxidoreductase.
The detection limits under bacterial culture and oyster sample con-ditions were 107 and 108 cfu·ml−1, respectively. However, the concen-tration of V. parahaemolyticus in shellfish can be considerably low(Fuenzalida et al., 2006; Garcia et al., 2009). To improve the sensitivityand detection limit, we performed a pre-enrichment experiment. Theresults showed that after 4 h of enrichment culture, the oyster samplewith an initial concentration of 100 cfu·ml−1 was detected by thecoupled enzyme system. The entire detection procedure (includingpre-enrichment and detection) was completed within 4.5 h, which isconsiderably shorter than that required by traditional detection
methods. Current molecular detection methods such as real-time poly-merase chain reaction (Nordstrom et al., 2007) and loop-mediated iso-thermal amplification (Nemoto et al., 2009) are rapid, accurate, andeasily standardized. However, these methods require complex proce-dures and expensive equipment that limit their practical application(Parida et al., 2005). Although it cannot strictly reflect the original bac-terial quantity through enrichment culture, the detection system pre-sented in this study is an attractive technology that can be appliedeasily at low cost. Further research on the purification, co-immobiliza-tion, recycling, and stability of bacterial luciferase and FMN:NADH oxi-doreductase should be performed to develop detection kits, portabledetection instruments, and biosensors.
The success of the phage-based detection system in this study de-pends on phage specificity. It is extremely important that the phagelyse a large number of food isolates of the target strain while not affect-ing non-target bacteria. Due to the specific recognition, the phage VPp1cannot infect all the species of V. parahaemolyticus from different envi-ronments (Table 1), which restricted the application of phage VPp1.However, this study is a foundational research to find out the feasibilityof NADH extraction by bacteriophage. Artificial methods to extendphage host range have been studied (Marzari et al., 1997; Heilpernand Waldor, 2003; Moradpour and Ghasemian, 2011). Phage cocktailsto control pathogenic bacteria have been used and they effectively over-come this disadvantage (O'Flynn et al., 2004; Tanji et al., 2004, 2005).Our lab has finished the genome sequence detection of VPp1 and thehost range could be extended by gene reconstruction in the furtherstudy.
Acknowledgments
This study was supported by the National Natural Science Founda-tion of China (No. 31071540), the Research Fund for the Doctoral Pro-gram of Higher Education of China (No. 20100132110013), and theEarmarked Fund for China Agriculture Research System (No. CARS-50). The authors declare no conflicts of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.mimet.2014.01.005.
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