Food Control 51 (2015) 31e36

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Development of a multiplex real-time PCR method for simultaneous

detection of Vibrio parahaemolyticus, Listeria monocytogenes and
Salmonella spp. in raw shrimp
Zhaohuan Zhang a, Lili Xiao a, Yang Lou a, Mengtong Jin a, Chao Liao a,
Pradeep K. Malakar a, d, Yingjie Pan a, b, c, Yong Zhao a, b, c, *
a

College of Food Science and Technology, Shanghai Ocean University, 999# Hu Cheng Huan Road, Shanghai, 201306, China
Laboratory of Quality & Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, 999# Hu Cheng
Huan Road, Shanghai, 201306, China
c
Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, 999# Hu Cheng Huan Road, Shanghai, 201306, China
d
Institute of Food Research, Norwich Research Park, Colney, Norwich, NR47UA, United Kingdom
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 18 June 2014
Received in revised form
26 October 2014
Accepted 4 November 2014
Available online 12 November 2014

Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. are important pathogens contaminating seafood in China. In this study, we developed an efcient multiplex real-time PCR for the
simultaneous detection of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in raw shrimp
without a prior enrichment step. In a test using 28 target and non-target strains only the targets were
detected and two calibration curves, for pure cultures and articially contaminated samples, were used
to evaluate the efciencies of this method. Amplication efciencies of this multiplex real-time PCR were
excellent in pure cultures and articially contaminated shrimps. The limits of detection in articially
contaminated shrimps were 112 CFU/g for V. parahaemolyticus, 158 CFU/g for L. monocytogenes and
103 CFU/g for Salmonella spp. We validated this multiplex real-time PCR method on 48 commercial
samples and the results were comparable to standard culture methods. This efcient multiplex real-time
PCR, where each test takes only 50 min after DNA extraction, is a useful tool for high-throughput surveillance of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in seafood products.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Multiplex real-time PCR
Raw shrimp
Vibrio parahaemolyticus
Listeria monocytogenes
Salmonella spp.

1. Introduction
Major outbreaks of foodborne disease have been documented
on every continent in the past decade (WHO, 2009). In China, 5021
foodborne disease outbreaks were reported by the National Foodborne Diseases Surveillance Network during 2001e2010, resulting
in 140,101 reported cases of illness and 1427 deaths. Microbial
pathogens were the causes of 40.9% of outbreaks and 56.4% of illnesses (Xu & Zhang, 2012).
Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella
spp. are major causes of foodborne illnesses worldwide (Coburn,
Grassl, & Finlay, 2007; Kathariou, 2002; Norhana, Poole, Deeth, &
Dykes, 2010; Su & Liu, 2007) and V. parahaemolyticus causes the
most cases of foodborne illnesses in China (Chen et al., 2010, Chen,

* Corresponding author. Tel./fax: 86 21 6190 0503.

E-mail address: yzhao@shou.edu.cn (Y. Zhao).
http://dx.doi.org/10.1016/j.foodcont.2014.11.007
0956-7135/ 2014 Elsevier Ltd. All rights reserved.

Liu, Fan, & Wang, 2008; Liu, Chen, Fan, Wang, 2006, Liu, Chen, Guo,
& Wang, 2008, Liu et al., 2005, Xu et al., 2012). Consumption of food
contaminated with pathogenic V. parahaemolyticus may lead to the
acute gastroenteritis with nausea, headache and low fever (Oliver &
Kaper, 2001; Su & Liu, 2007). Salmonellosis ranks second in China
and constitutes 10e20% of bacterial foodborne illness outbreaks
(Chen et al., 2008; Liu, Chen, Wang, & Ji, 2004, 2006, 2008). In
China, foodborne outbreaks caused by L. monocytogenes is minor
but this pathogen is widely distributed in many food materials
(Farber, Ross, & Harwig, 1996; Kathariou, 2002; McLauchlin,
Mitchell, Smerdon, & Jewell, 2004). Therefore the potential for
listeriosis is high in China and is a major concern for public health
authorities (CFSA, 2014; Chen et al., 2013; Chen et al., 2014; Yan
et al., 2010; Yu & Jiang, 2014).
Seafood products are increasingly popular among consumers
worldwide (Amagliani, Brandi, & Schiavano, 2012). In China, seafood production and consumption has grown progressively during
the past ten years, and 3,033,000 tons of seafood were produced in

32

Z. Zhang et al. / Food Control 51 (2015) 31e36

2012 (State Statistical Bureau, 2013). V. parahaemolyticus,

L. monocytogenes and Salmonella spp. are important hazards in
seafood products (Akiyama, Khan, Cheng, & Stefanova, 2011;
Amagliani et al., 2012; Chen et al. 2012; Costa Sobrinho, Destro,
Franco, & Landgraf, 2011; Embarek, 1994; Fallah, Saei-Dehkordi, &
 n,
Mahzounieh, 2013; Gonz
alez, Vitas, Dez-Leturia, & Garca-Jalo
2013; Ponce, Khan, Cheng, Summage-West, & Cerniglia, 2008;
Rocourt, Jacquet, & Reilly, 2000) and the development of a rapid,
accurate and internationally accepted method for detection of
these hazards in seafood is important from a public health
perspective.
The gold-standard for the detection of these three pathogens,
based on cultural enrichment, selective plating and biochemical
conrmation, is labor-intensive, complicated and time-consuming
(Garrido et al., 2013; Kim, Lee, Lee, & Cho, 2012; Ma et al., 2014).
Polymerase chain reaction (PCR), which is less labor-intensive, is
suitable for detection of a variety of pathogens in food (Feng, 2007)
and a multiplex PCR can be used for simultaneous detection of
various target pathogens in a single reaction. However, both PCR
and multiplex PCR methods are still time-consuming, as they
require an electrophoresis step to conrm the amplication products. Automated real-time PCR was developed as a rapid detection
method, without need for an electrophoresis step after amplica n, & Aznar, 2011; Ma et al., 2014), with the
tion (Elizaquvel, Gabaldo
added advantage of reducing the risk of cross contamination.
Multiplex real-time PCR can simultaneously and directly amplify
more than two gene sequences in the same reaction, and has been
used for detection of many pathogens in food (Elizaquvel & Aznar,
2008; Garrido et al., 2013; Hyeon, Park, Choi, Holt, & Seo, 2010; Kim
et al., 2012; Ma et al., 2014; Wolffs, Glencross, Norling, & Grifths,
2007).
In this study, we developed a multiplex real-time PCR method
for rapid and simultaneous detection of V. parahaemolyticus, L.
monocytogenes and Salmonella spp. in raw shrimp. To our knowledge, this is the rst study to deal with the simultaneous detection
of these three pathogens in raw shrimp by using multiplex realtime PCR. We hope this detection method can provide an effective technical support for improving public health in China and the
rest of the world.
2. Materials and methods
2.1. Bacterial strains and culture conditions
The 28 bacterial strains used in this study are listed in Table 1. V.
parahaemolyticus strains were grown in tryptic soy broth (TSB;
Beijing Land Bridge Technology Company Ltd., Beijing, China)
supplemented with 3.0% NaCl and incubated at 37  C for 18 h. Other
strains were grown in TSB at 37  C for 18 h to 20 h.

Table 1
Specicity of the multiplex real-time PCR assay for different bacterial strains.
Bacterial species

Vibrio parahaemolyticus

Listeria monocytogenes

Salmonella spp.
Salmonella enterica
Salmonella Paratyphi B
Salmonella typhimurium
Other stains
Vibrio anguillarum
Vibrio uvialis
Vibrio vulnicus
Listeria innocua
Listeria innocua
Listeria innocua
Listeria welshimeri
Listeria welshimeri
Escherichia coli O157:H7
Staphylococcus aureus
Pseudomonas fragi
Rahnella aquatilis
Megacoccus caseolyticus
Staphylococcus saprophyticus
Hafnia alvei

Strain

Multiplex real-time
PCR results
tlh

hlyA

orgC

ATCC 33847
ATCC 17802
VP6 (wild type)
VP18 (wild type)
VP57 (wild type)
ATCC 19115
ATCC 19112
ATCC 19114
ATCC 19116
ATCC 19118

e
e
e
e
e

e
e
e
e
e

e
e
e
e
e
e
e
e
e
e

CMCC 50041
CMCC 50094
CICC 21484

e
e
e

e
e
e

CICC 10474
CGMCC 1.1611
MCCC1H0006
No.11-yl (wild type)
No.47-E1 (wild type)
ATCC 33091
ATCC 43548
ATCC 43550
ATCC 43889
AB 91093
No.20-L2 (wild type)
No.23-P4 (wild type)
No.26-R3 (wild type)
No.27-R4 (wild type)
No.31-T2 (wild type)

e
e
e
e
e
e
e
e
e
e
e
e
e
e
e

e
e
e
e
e
e
e
e
e
e
e
e
e
e
e

e
e
e
e
e
e
e
e
e
e
e
e
e
e
e

ATCC, American Type Culture Collection, USA; CMCC, China Medical Culture
Collection, China; CGMCC, China General Microbiological Culture Collection Center,
China; China Center of Industrial Culture Collection. /indicates positive/negative real-time PCR signal.

where the incubation time in lysozyme was increased to 1 h and the

incubation time in proteinase K was increased to 2 h (Ye et al. 2013).

2.4. Primers and probes used for multiplex real-time PCR

Primers and probes were synthesized by Invitrogen Corp
(Shanghai, China). Primers and probes used in this study for the
simultaneous detection of V. parahaemolyticus, L. monocytogenes
and Salmonella spp. are listed in Table 2.
To determine the specicity of the primers and probes, multiplex real-time PCR as described below was done with the 28 strains
in Table 1, including 13 target and 15 non-target strains. These
experiments were done twice.

2.2. Shrimp samples

2.5. Multiplex real-time PCR
Shrimp samples (Penaeus vannamei) were purchased from a
local supermarket. These samples were conrmed to be free of
V. parahaemolyticus, L. monocytogenes and Salmonella spp. by using
standard culture methods (ISO, 1998, 2002, 2007) as described in
Fig. 1. The presence of the natural microbiota present in shrimp was
shown by spreading on tryptic soy agar (TSA; Beijing Land Bridge
Technology Company Ltd., Beijing, China).
2.3. DNA extraction
Bacterial DNA was extracted using the TIANamp Bacteria DNA
Kit (Tiangen Biotech Beijing Co., Ltd., China) according to the
manufacturer's instruction. This extraction method was modied,

Multiplex real-time PCR was carried out in a nal volume of

20 mL: 2 mL of 10  PCR Buffer (Invitrogen, USA), 1.2 mL of 50 mM
MgSO4 (Invitrogen, USA), 0.5 mL of 10 mM dNTPs mix (Invitrogen,
USA), 0.2 mL of Taq DNA polymerase (5 U/mL) (Invitrogen, USA),
0.5 mL 10 mM primer and 0.2 mL of 10 mM probe were used for each
strains, 1 mL of DNA were used as template.
A 7500 Fast real-time PCR system (Applied Biosystems, Foster
City, CA, USA) was used with the following thermal prole: initial
denaturation of genomic DNA at 95  C for 2 min, followed by 40
cycles of denaturation at 95  C for 15 s, and annealing at 60  C for
1 min. Analysis of the results was performed using 7500 Software
v2.0.6.

Z. Zhang et al. / Food Control 51 (2015) 31e36

33

Fig. 1. Schematic representation of standard culture methods compared with multiplex real-time PCR methods.

2.6. Standard curves and amplication efciency

Two calibration curves were constructed to determine amplication efciency of the multiplex real-time PCR method.
A standard curve was obtained by using genomic DNA from
V. parahaemolyticus ATCC 33847, L. monocytogenes ATCC 19115 and
Salmonella enterica CMCC 50041 extracted from serial dilutions of a
bacterial suspension. The culture was diluted in sterile peptone
water (PW; 0.85% NaCl, 0.1% peptone) to obtain concentrations
ranging from 101 to 108 CFU/ml.
The other standard curve was obtained from bacterial DNA
extracted from raw shrimp dilutions previously inoculated with 10fold serially diluted concentrations of three pathogens: A 25-g
portion of raw shrimp was aseptically weighed into a sterile plastic bag, and 225 ml PW was homogenized for 2 min in a stomacher
400 to obtain a 10-fold shrimp dilutions. Eight plastic bags within
shrimp homogenate were inoculated with 10-fold serially diluted
suspensions of V. parahaemolyticus ATCC 33847, L. monocytogenes
ATCC 19115 and S. enterica CMCC 50041 respectively. The nal
concentrations of bacteria in shrimp dilutions were range from 102
to 108 CFU/g.
A direct-plating procedure was used for the enumeration of
V. parahaemolyticus, L. monocytogenes and S. enterica in pure cultures and samples, thiosulfate-citrate-bile salts-sucrose agar culture medium (TCBS; Beijing Land Bridge Technology Company Ltd.,
Beijing, China) for V. parahaemolyticus, PALCAM agar (Beijing Land
Bridge Technology Company Ltd., Beijing, China) for
L. monocytogenes and Bismuth Sulte agar (BS; Beijing Land Bridge
Technology Company Ltd., Beijing, China) for Salmonella spp., which
were incubated for 18 h, 24 h and 36 h at 37  C respectively. The

different bacterial concentrations (log CFU/g) were plotted against

the corresponding CT values. The slope of the linear relationship of
this curve was used to determine the amplication efciency (E) by
 et al.,
applying the following equation: E 101/slope  1 (Mace
2013).
2.7. Evaluation of the limit of detection (LOD) by multiplex realtime PCR
To determine the LOD in practical samples, three groups of raw
shrimp (20 1 g per sample) with 5 samples each were prepared as
follow: each shrimp was simultaneously inoculated serial dilutions
of V. parahaemolyticus, L. monocytogenes and Salmonella spp. The
nal contaminations of three pathogens in shrimp were 101, 102 or
103 CFU/g and listed in Table 3. Bacteria strains used for the LOD
experiment were V. parahaemolyticus ATCC 33847, L. monocytogenes
ATCC 19115 and S. enterica CMCC 50041.
Then each sample was added in 180 ml PW and homogenized
for 2 min in a stomacher 400. DNA was extracted from each homogenate and then used in subsequent multiplex real-time PCR as
described in Section 2.3 and 2.5. This experiment was repeated
twice independently.
2.8. Detection of naturally contaminated samples
A total of 48 raw shrimp samples were purchased from 3 main
wholesale markets dealing with aquatic products in Shanghai and
these samples were analyzed by multiplex real-time PCR method
and standard culture methods for V. parahaemolyticus,
L. monocytogenes and Salmonella spp. (ISO, 1998, 2002, 2007).

Table 2
Primer and probe sequences used for multiplex real-time PCR.
Bacteria

Gene

Primers/probes

Product
size (bp)

Reference

V. parahaemolyticus

tlh

208

Nordstrom et al., 2007

L. monocytogenes

hlyA

137

Omiccioli et al., 2009

Salmonella spp.

orgC

50 -ACTCAACACAAGAAGAGATCGACAA-30
50 -GATGAGCGGTTGATGTCCAA-30
50 -FAM-CGCTCGCGTTCACGAAACCGT-BHQ1-30
50 -ACTTCGGCGCAATCAGTGA-30
50 -TTGCAACTGCTCTTTAGTAACAGCTT-30 50 -ROX-TGAACCTACAAGACCTTCCAGATTTTTCGGC-BHQ1-30
50 -CTTTATGATGCATTCTACCAACGACTG-30
50 -CCGAATCACCACTGTTAGGA-30
50 -HEX-CGCTTCCTGAGTCAGCCTCTTCTGAAACG-BHQ1-30

121

Day et al., 2009

34

Z. Zhang et al. / Food Control 51 (2015) 31e36

Table 3
The limit of detection (LOD) of the multiplex real-time PCR in articially contaminated shrimps.
Target microorganism

Concentration
of strains
(CFU/g)

Multiplex real-time
PCR results
tlh

hlyA

orgC

V. parahaemolyticus, L. monocytogenes,
Salmonella spp.
V. parahaemolyticus, L. monocytogenes,
Salmonella spp.
V. parahaemolyticus, L. monocytogenes,
Salmonella spp.

101

103

10

/indicates positive/negative multiplex real-time PCR signal.

A 25-g portion of raw shrimp was aseptically weighed into a sterile

plastic bag, and 225 ml of PW was homogenized for 2 min in a
stomacher 400. After homogenizing, one milliliter of supernatants
was transferred to new tubes and centrifuged at 12,000 rpm for
10 min at 4  C to DNA extraction prepared for subsequent multiplex
real-time PCR assay.
Other 25-g portion of raw shrimp was homogenized with
Alkaline Peptone Water (APW; Beijing Land Bridge Technology
Company Ltd., Beijing, China) and incubated overnight at 37  C, and
then streaked onto the TCBS agar, PALCAM agar and BS agar and
incubated for 18 h, 24 h and 36 h at 37  C respectively. Three typical
colonies from each plate were conrmed by additional biochemical
conrmation where the presumptive V. parahaemolyticus and Salmonella colonies were validated using the API 20E test (Biorieux), Presumptive L. monocytogenes colonies were conrmed
Me
rieux) (Fig. 1).
with the API 10300 test (BioMe
3. Results
3.1. Specicity of primers and probes for multiplex real-time PCR
The specicity of the primers and probes was tested using 13
target strains as positive controls and 15 bacterial strains (Table 1)
as negative controls. As shown in Table 1, the multiplex real-time
PCR assays did not show any positive signal for all non-target
strains, and only observed with target strains. That indicated the
high specicity of the primers and probes.
3.2. Standard curve and amplication efciency
Two different calibration curves were constructed for the efciency
evaluation of the multiplex real-time PCR method (Fig. 2). By using the
genomic DNA extracts from the pure cultures, the slopes of the linear
regression curve were 3.175 for V. parahaemolyticus, 3.1073 for
L. monocytogenes and 3.129 for Salmonella spp. Amplication efciencies for V. parahaemolyticus, L. monocytogenes and Salmonella spp.
were: 107%,112% and 109% respectively, with R-square values of 0.995,
0.998, and 0.998 respectively.
On the articially contaminated shrimp model, the slopes
were 3.175 for V. parahaemolyticus, 3.082 for L. monocytogenes
and 3.264 for Salmonella spp. Amplication efciencies for
V. parahaemolyticus, L. monocytogenes and Salmonella spp. were:
104%, 111% and 102% respectively, with R-square value of 0.995,
0.998, and 0.998 respectively.
3.3. Evaluation of the LOD in articially contaminated shrimp
samples by multiplex real-time PCR
The evaluation of the LOD was done in three groups with 5
samples each, which were articially contaminated with three
pathogens, and the procedures were described in Section 2.7.

Fig. 2. Standard curves for multiplex real-time PCR in pure culture (C) or in articially
contaminated shrimp ( ). A: Standard curves for Vibrio parahaemolyticus. B: Standard
curves for Listeria monocytogenes. C: Standard curves for Salmonella spp. Viable counts
were determined using selective medium (TCBS, PALCAM and BS).

The nal levels in the three groups were 101, 102 and 103 CFU/g
and only the group with 101 CFU/g was negative for the multiplex
real-time PCR (Table 3). The resultant LOD values of the multiplex
real-time PCR were 112 CFU/g for V. parahaemolyticus, 158 CFU/g for
L. monocytogenes and 103 CFU/g for Salmonella spp.
3.4. Comparison of multiplex real-time PCR and standard culture
methods for detection of three pathogens in naturally contaminated
samples
A total of 48 raw shrimp samples were purchased from 3 major
seafood wholesale markets in Shanghai and these samples were

Z. Zhang et al. / Food Control 51 (2015) 31e36

analyzed by the multiplex real-time PCR method and by standard

culture methods. Forty-ve out of all samples were positive for
V. parahaemolyticus using the multiplex real-time PCR method and
the standard method. Twelve samples were positive for
L. monocytogenes and twenty-six were positive for Salmonella spp.
by both methods.
4. Discussion
Microbiological analysis of foodstuffs is a prerequisite for microbial safety management. Developing a rapid, accurate and
internationally accepted method for detection of pathogens in food
is challenging and presently multiplex real-time PCR has become a
popular tool for the detection of pathogenic bacteria in food
(Elizaquvel & Aznar, 2008; Garrido et al., 2013; Hyeon et al., 2010;
Kim et al., 2012; Ma et al., 2014; Wolffs et al., 2007). In this study,
we developed a multiplex real-time PCR assay for the simultaneous
detection of V. parahaemolyticus, L. monocytogenes and Salmonella
spp. using tlh, hlyA and orgC as target genes. This method successfully detected these three pathogens in articially and naturally
contaminated seafood samples.
The primers and probes used in this study has been reported in
previous real-time PCR research for the detection of
V. parahaemolyticus (Nordstrom, Vickery, Blackstone, Murray, &
DePaola, 2007), L. monocytogenes (Omiccioli, Amagliani, Brandi,
Magnani, 2009) and Salmonella spp. (Day, Basavanna, & Sharma,
2009). However this is the rst study to integrate these primers
and probes into a PCR reaction for simultaneous identication of
these three pathogens. Even though these primers and probes have
a high specicity (Day et al., 2009; Nordstrom et al., 2007; Omiccioli
et al., 2009), we were unsure if the integration of the primer sets
and probes with relabeling of different uorophores and the
adjustment of the PCR system would retain these specicity. We
reconrmed the specicity of the integrated primers and probes on
28 strains (Table 1) where only the targets were detected and no
false positives occurred. Additionally, the amplication efciencies
of the new PCR assay were tested by constructing two different
calibration curves. Whether in pure culture or in articially
contaminated shrimp, this multiplex real-time PCR system
possessed excellent efciencies for the amplication of each target
(Fig. 2).
A preliminary sterilization step is usually used to adjust for
background bacterial ora in articially contaminated food samples
 et al., 2013; Ye et al., 2013). However, in this study we chose
(Mace
raw shrimp as the matrix without any pretreatments. The results
showed that the PCR reaction was free from interference of the
complex natural microbiota, and we obtained high specicity and
amplication efciencies (Fig. 2 and Table 3). We are condent that
the multiplex real-time PCR method developed in this manuscript
can be tuned to detect multiple pathogens in other food samples.
In the present study, the limit of detection of this multiplex realtime PCR method was 102 CFU/g in articially contaminated shrimp
(V. parahaemolyticus, 112 CFU/g; L. monocytogenes, 158 CFU/g; Salmonella spp., 103 CFU/g). Comparably, these LOD values are
approximately one order of magnitude lower than most multiplex
real-time PCR methods applied to food, without prior enrichment.
Elizaquvel & Aznar (2008) reported a multiplex real-time PCR
assay allowed detection of 103 CFU/g for Escherichia coli O157:H7,
Salmonella spp. and Staphylococcus aureus on vegetables. Hyeon
et al. (2010) obtained a detection limit of 103 CFU/ml for Salmonella and Cronobacter in powdered infant formula. Bottari,
Agrimonti, Gatti, Neviani, and Marmiroli (2013) obtained a sensitivity of 102e103 copies/reaction for lactic acid bacteria in natural
whey starters. Lower detection levels between 100e102 CFU/ml
have been reported recently, where the enrichment step is

35

increased by several hours, i.e., Vibrio species in seafood (Kim et al.,

2012), Salmonella spp. and L. monocytogenes in food and environmental samples (Garrido et al., 2013), L. monocytogenes, Salmonella
spp. and E. coli O157:H7 in ready-to-eat products (Kotzekidou,
2013). If the initial contamination levels of samples are too low,
we suggest adding an enrichment step prior to the PCR assay as the
growth rates of these pathogens are high at 37  C (Carrasco, Rosal,
Racero, & Garca-Gimeno, 2012; Cornu, Kalmokoff, & Flandrois,
2002; Yoon et al., 2008) with relatively short lag times.
When the multiplex real-time PCR assay was applied to 48
commercial samples and compared with standard culture methods,
both methods adequately detected the bacterial loads in the samples. However, as depicted in Fig. 1, the standard culture methods
are labor-intensive, complicated and time-consuming, and this
multiplex real-time PCR method was simpler and took only about
50 min after DNA extraction. By comparison between two methods,
the multiplex real-time PCR method is not only accurate and
effective, but also more rapid.
In conclusion, a rapid and simultaneous multiplex real-time PCR
was developed for the detection of V. parahaemolyticus,
L. monocytogenes and Salmonella spp. in raw shrimp. The method
showed high sensitivity, specicity, accuracy and achieved a low
limit of detection (102 CFU/g for each pathogen) without prior
enrichment. The savings in time (approximately 50 min after DNA
extraction) when compared to standard methods is a major
advantage. This new multiplex real-time PCR method is a valid
assay for high-throughput surveillance of these three pathogens in
seafood products and a tool for improving public health in China.
Acknowledgments
This research was supported by the National Natural Science
Foundation of China (31271870), the project of Science and Technology Commission of Shanghai Municipality (14DZ1205100,
14320502100, 12391901300), Key Project of Shanghai Agriculture
Prosperity through Science and Technology (Grant: 2014, 3-5),
Cross-Discipline Project (B5201120040). Pradeep Malakar was
supported by the Biotechnology and Biological Sciences Research
Council (BBSRC) of the UK.
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