Simultaneous detection of six food-borne pathogens by multiplex PCR with a GeXP analyzer

Accepted Manuscript Simultaneous Detection of six food-borne pathogens by multiplex-PCR with a GeXP analyzer Beili Zhou, Jinwen Xiao, Shengfeng Liu, Jun Yang, Yu Wang, Fuping Nie, Qing Zhou, Yingguo Li, Guohua Zhao PII:

S0956-7135(12)00646-9

DOI:

10.1016/j.foodcont.2012.11.044

Reference:

JFCO 3047

To appear in:

Food Control

Received Date: 11 July 2012 Revised Date:

21 November 2012

Accepted Date: 27 November 2012

Please cite this article as: ZhouB., XiaoJ., LiuS., YangJ., WangY., NieF., ZhouQ., LiY. & ZhaoG., Simultaneous Detection of six food-borne pathogens by multiplex-PCR with a GeXP analyzer, Food Control (2013), doi: 10.1016/j.foodcont.2012.11.044. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Simultaneous Detection of six food-borne pathogens by multiplex-PCR with a

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GeXP analyzer

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Beili Zhoua,b,1, Jinwen Xiaob,c,1, Shengfeng Liub,c, Jun Yangb,c, Yu Wangb,c, Fuping

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Nieb,c, Qing Zhoub,c, Yingguo Lib,c, Guohua Zhaoa,d,*

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a

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400020, PR China

College of Food Science, Southwest University, Chongqing 400715, PR China

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Chongqing Entry-Exit Inspection and Quarantine Bureau of China, Chongqing

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PR China

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400715, PR China

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These authors contributed equally to this work.

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Corresponding author

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College of Food Science

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Southwest University

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Key Laboratory of Food Processing and Technology of Chongqing, Chongqing

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Chongqing Import and Export Food Safety Engineering Center, Chongqing 400020,

# 1 Tiansheng Road, Chongqing, 400715 PR China

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Tel.: +86 23 68 25 19 02

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Fax: +86 68 25 19 47

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E-mail address: [email protected] (G. Zhao)

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Abstract A novel application of GeXP analyzer was developed for simultaneous detection

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of six pathogens associated with food poisoning outbreaks, including Salmonella

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enterica, Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus,

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Shigella spp., and Campylobacter jejuni. Chimeric primers containing both microbe-

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and pMD19-specific sequences were fused to universal sequences resulted in PCR

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products with intended sizes. The PCR products were separated by capillary

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electrophoresis and identified by using fluorescence spectrophotometry. Plasmid

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pMD19-T was added to each reaction as positive control to ascertain the PCR steps

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and data analysis step of the assay. The results indicate that the GeXP method is both

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specific and sensitive for the detection of all six food-borne pathogens in a single

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reaction-without any enrichment step. In conclusion, the GeXP-PCR assay is a rapid,

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sensitive and high throughput method for parallel analysis of food-borne pathogens.

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Keywords: food-borne pathogens; GenomeLab Gene Expression Profiler (GeXP);

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multiplex PCR; parallel analysis

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1. Introduction Illnesses resulting from the consumption of foods contaminated with pathogens

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and/or their toxins have a wide range of economic and public health impact

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worldwide (Motarjemi & Käferstein, 1999). Therefore, it is important to develop and

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validate methods for the rapid detection of biological contaminants in foods.

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Conventional detection methods for pathogenic bacteria rely primarily on direct

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plating methods and biochemical tests, which are time-consuming and labor-intensive

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(Kim et al., 2007). Consequently, a variety of researchers have tried to develop rapid

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and sensitive analytical methods for the detection of pathogenic bacteria. The

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polymerase chain reaction (PCR) is the most commonly employed molecular tool for

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the detection of pathogens. It allows the target gene of specific bacteria present at

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extremely low level to be identified after exponential amplification (Tang, Procop, &

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Persinga, 1997). However, the conventional PCR methods require agarose gel

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analysis to detect PCR products, which was unsuitable for high-throughput analysis

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and have a lower sensitivity than real time PCR (Perry et al., 2007). Compared to

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conventional PCR, the Taqman probe-based real-time PCR has some advantages,

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including potential for quantification, higher sensitivity, and minimal crosscontamination potential, and is widely used in various applications including pathogen diagnosis (Rodríguez-Lázaro et al., 2005; Amoroso et al., 2011). However, the limited

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availability of spectrally distinct fluorescent tags has impeded analysis using real-time

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PCR of more than four target pathogens per sample in a single run (Grace et al., 2003).

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Another popular method, the DNA microarray analysis, offers an efficient approach to 3

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detect microbial pathogens. In recent times, it has been available for the rapid

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detection and identification of 170 strains of bacteria belonging to 11 genera or

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sequence-specific identification of up to 18 pathogenic microorganisms (Jin et al.,

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2005; Wilson et al., 2002). Although the development of microarray platform has

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made it capable of high throughput analysis, the method has a disadvantage in the

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accuracy of identification. DNA microarray technology is based on the hybridization

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of probes to target genes, and therefore, non-specific hybridization is inevitable, and

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differences in the melting temperatures of probes and targets reduce the resolution of

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the method (Shin, Hwang, Oh, Doh, & Jung, 2010). Moreover, the limitations in

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dynamic range leading to saturation at higher intensity levels and lack of standard

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procedures for signal/noise optimization have seriously reduced the utility of

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microarray technology and impeded the subsequent development of downstream

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applications (Rai, Kamath, Gerald, & Fleisher, 2009).

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The GenomeLab Gene Expression Profiler (GeXP) genetic analysis system was

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originally designed to allow for a high throughput, robust and differential assessment

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of a multiplex gene expression profiling analysis (Rai, Kamath, Gerald, & Fleisher,

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2009). The GeXP-PCR detection can combine patented XP-PCR priming strategy

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with capillary electrophoretic separation, resulting in a high level of specificity, sensitivity and high throughput capacity. With the GeXP, up to 35 genes can be easily

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multiplexed in a single reaction. The major advantages of this method include high

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performance, high multiplexing capability, and the ability to process samples in

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parallel. These advantages have further widened its areas of application. Recently, the

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GeXP analyzer has been used in simultaneous detection and identification of up to 68

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unique varicella zoster virus gene transcripts (Nagel, Gilden, Shade, Gao, & Cohrs,

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2009) or genotyping of 11 human papillomavirus in a single reaction (Yang et al.,

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2012). However, there has been no report on the detection or identification of

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pathogenic bacteria using the GeXP analyzer.

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Salmonella is the leading cause of food-borne illness, which is a significant risk to

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public health (Taskila, Tuomola, & Ojamo, 2012). Worldwide, Escherichia coli

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O157:H7 is considered as a leading cause of diarrhea, hemorrhagic colitis and

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hemolytic-uremic syndrome (Besser, Lett, Weber, & Doyle, 1993). Listeria

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monocytogenes is associated with listeriosis, a severe disease with high morbidity and

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mortality rates (Todd & Notermans, 2011). Staphylococcus aureus is a major pathogen

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that causes serious infections in humans, including pneumonia, septicemia and

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endocarditis (McClelland et al., 1999). In developing countries, shigellosis caused by

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Shigella spp. is a prevalent diarrhea disease (Carlson, Thornton, DuPont, West, &

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Mathewson, 1983). In recent times, Campylobacter jejuni has received increasing

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attention as a predominant cause of bacterial food-borne enteritis in many countries

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(Piddock, Ricci, Stanley, & Jones, 2000).

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The objective of this study was to develop a rapid, sensitive and high throughput

pathogenic bacteria diagnostic technique using GeXP-based multiplex PCR assay

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(GeXP-PCR) for simultaneous detection of six food-borne pathogens, namely

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Salmonella enterica, Shigella spp., Listeria monocytogenes, Campylobacter jejuni,

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Staphylococcus aureus and Escherichia coli O157:H7.

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2. Materials and methods

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2.1. Bacterial strains and their cultivation The strains used for specificity testing are listed in Table 1. For the identification of

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six food-borne pathogens and the sensitivity of the GeXP assay experiments, the

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following strains were used: Salmonella enterica serotype Enteritidis ATCC13076,

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Escherichia coli O157:H7 ATCC8739, Listeria monocytogenes ATCC15313,

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Staphylococcus aureus ATCC6538, Campylobacter jejuni ATCC33291, and Shigella

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flexneri serotype 2b ATCC12022. Campylobacter jejuni was microaerobically

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incubated in brucella broth (BB; Difco laboratories, Detroit, MI) at 42°C for 48 h.

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Clostridium perfringens was anaerobically grown in tryptic soy broth (TSB; Difco

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laboratories, Detroit, MI) at 37°C for 24 h. The other bacterial strains were grown in

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tryptic soy broth (TSB) or brain-heart-infusion (BHI; Oxoid Ltd., Basingstoke, UK) at

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37°C for 24 h.

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2.2. Preparation of positive-control plasmid pMD-19

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The pMD19-T vector (TaKaRa, Japan) was transformed in competent Escherichia

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coli DH5α(Life Technologies Inc., Gaithersburg, MD.) and the cell suspension was

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added to the Luria broth. After being incubated overnight at 37°C, the empty plasmid

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was extracted using an E.Z.N.A Plasmid Extraction Kit (Omega Biotek Inc., Guangzhou, China). The plasmid DNA obtained was added to each reaction during

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the multiplex PCR step as positive control.

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2.3. GeXP-PCR Primer Design

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Two types of primers were present in the reaction: 1) Chimeric primers containing a

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gene-specific sequence with a universal tag at the 5’ end; 2) Universal primers that

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have the same sequence as the universal tags used in the chimeric primers. Six pairs

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of chimeric primers, one pair of positive-control primers, and one pair of universal

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primers (UWD-F/UEV-R) were designed for this assay. Six pairs of chimeric primers

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were designed according to the conserved regions of the target bacterial gene segment

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using the GenomeLab GeXP eXpress Profiler software (Beckman Coulter, Fullerton,

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CA). One pair of primers for the detection of plasmid pMD19-T gene that was used as

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positive control was also design. The primer sequences, the target regions, and the

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size of the resulting amplicons are listed in Table 2. The forward universal primer was

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covalently labeled with Cy5 fluorescent dye at the 5’ end and yielded signals that

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corresponded to the amount of product in the multiplex reaction. All chimeric primers

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and the universal primers were obtained from Takara Corporation (Dalian, China).

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2.4. Genomic DNA extraction and GeXP multiplex PCR

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Genomic DNAs of all strains were extracted with an E.Z.N.A Bacterial DNA

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Isolation Kit (Omega Biotek Inc., Guangzhou, China) according to manufacturer's

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procedures. PCR amplification of DNA from bacteria was performed using the Veriti

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96-Well Thermal Cycler (Applied Biosystems, Foster City, CA). PCR amplification

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was performed in a 20 µL reaction volume containing 2 µL (200-500 ng) of bacterial genomic DNA, 2 µL (200-500 ng) of plasmid pMD-19 DNA, 10 µM of each of the

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forward universal primer and reverse universal primer, 1 µM of each of

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the forward and reverse chimeric primers of each targeted bacteria, 0.5 µM of each of

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the pMD-19 forward chimeric primer and reverse pMD-19 chimeric primer, 3.0 mM

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of MgCl2 (Promega, Madison, WI), 0.15 U of Taq DNA polymerase (Promega,

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Madison, WI), 0.5 mM of each dNTP (Promega, Madison, WI), and 1×PCR Master

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Mix buffer (GenomeLab GeXP Start Kit; Beckman Coulter, Fullerton, CA)

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containing 10 mM of HCl and 50 mM of KCl. In addition, the plasmid pMD-19 DNA

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was added directly to the PCR mixture as positive control. Nuclease-free sterile

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distilled water (Invitrogen, Paisley, UK) was used as negative control throughout the

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assay. The amplification procedure consisted of two steps with different annealing

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temperatures: step 1: 12 PCR cycles of DNA denaturation at 94°C for 45 s, primer

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annealing at 52°C for 45 s, and DNA extension at 72°C for 30 s; step 2: 20 cycles at

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94°C for 45 s, 60°C for 45 s, and 72°C for 30 s. A part of the amplified sample was

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analyzed using 2.0% agarose gel electrophoresis. DNA bands were stained with

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ethidium bromide (EB) and photographed using the BIO-BEST 200E image system

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(SIM, HK, China).

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2.5. GeXP multiplex data analysis

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The GeXP system (Beckman Coulter, Fullerton, CA) was used to separate the PCR

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products and analyze the data according to the manufacturer’s instructions. The

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capillary array was pre-heated to 50 °C for 15 min before analysis. 1µL of the

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undiluted PCR product was added to a 37.75 µL of sample loading solution along with 0.25 µL of DNA size standard-400 (GenomeLab GeXP Start Kit; Beckman

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Coulter, Fullerton, CA). The PCR products were separated based on size using

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high-resolution capillary gel electrophoresis, presented as separated peaks in the

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electropherogram and identified in terms of their intended sizes. The data can be

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exported from the GeXP Express Analysis module (Beckman Coulter, Fullerton, CA)

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as a tab-delimited file for further statistical analysis. The presence of a target pathogen

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was considered in the initial template when the dye signal of the corresponding

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microbe-specific product was greater than 2000 arbitrary units (A.U.).

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2.6. Specificity of the GeXP-PCR assay

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To assess the specificity of the GeXP-PCR assay, cultures of 34 target and

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non-target bacterial strains (Table 1) were prepared. The purity of the genomic DNA

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was assessed by determining the A260/A280 ratio using a spectrophotometer

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(BioMate 3; ThermoSpectronic, Rochester, NY). The GeXP-PCR was performed

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individually for each of DNA samples of 34 strains in a multiplex primer system using

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the experimental conditions described above. The inclusivity and exclusivity were

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calculated according to the Microval protocol (Anonymous, 2002). Inclusivity is the

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ability of the PCR method to detect the target analyte from a wide range of

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strains. Inclusivity is defined as the percentage of target DNA samples that gave a

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correct positive signal. Exclusivity is defined as the percentage of non-target DNA

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samples that gave a correct negative signal.

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2.7. The detection limits of the GeXP-PCR assay

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Each target pathogen was cultured to obtain an approximate cell concentration in

the range of 108 to 109 CFU/mL. The cell suspension was serially diluted 10-fold with

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0.9% (w/v) NaCl to result in a cell concentration ranging from 106 to 100 CFU/mL.

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The concentration of each bacterium (4.2 × 108 CFU/mL of Salmonella, 9.3 × 108

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CFU/mL of E. coli O157:H7, 3.1 × 108 CFU/mL of L. monocytogenes, 2.7 × 108

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CFU/mL of S. aureus, 8.5 × 108 CFU/mL of Shigella spp., and 6.6 × 108 CFU/mL

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of C. jejuni) was determined by surface plating (0.1 mL) of the appropriate dilutions

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onto corresponding agar plates. The Campylobacter jejuni dilution was inoculated

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onto surface-dried charcoal cefoperazone deoxycholate agar (CCDA; Oxoid Ltd.,

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Basingstoke, UK) plate and the plate was subsequently microaerobically incubated at

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37°C for 48 h to count the colonies. For others, the number of bacteria in the culture

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was estimated by standard plate count. 200 µL of 6 bacteria suspensions with the same

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dilution were mixed to obtain the 1200 µL (200 µL × 6) bacteria mixture. The

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mixture was processed for DNA extraction and the DNA obtained was added directly

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to the PCR mixture. GeXP-PCR was performed using the experimental conditions as

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described above. After amplification, 1 µL of each Cy5-labeled PCR product was

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separated via GeXP capillary electrophoresis and detected by fluorescence

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spectrophotometry. The experiment was run in triplicate for each concentration

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ranging from 100 to 106 CFU/mL. The detection limit was determined as the lowest

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concentration of bacterial dilution, at which the positive result was reproducibly

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detected in all of the replicates.

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3. Results

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3.1. Identification of food-borne pathogens Six food-borne pathogens, including Salmonella enterica, Escherichia coli

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O157:H7, Listeria monocytogenes, Shigella spp., Staphylococcus aureus and

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Campylobacter jejuni were detected simultaneously via the GeXP multiplex PCR

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assay using multiplex primer set (Fig. 1 Panel (A)). No PCR product corresponding

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with target microorganism was detected in negative control using the multiplex primer

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set (Fig. 1 Panel (B)). PCR products corresponding with the positive-control plasmid

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pMD19-T gene were detected from both negative control and pure cultures of six

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pathogens.

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3.2. Specificity of the GeXP-PCR Assay

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The specificity of the GeXP-PCR assay was examined by isolating genomic DNA

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from 34 target and non-target bacterial strains. As shown in Fig. 2, amplification

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products of the expected sizes were obtained by PCR on six representative bacterial

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strains. All six Salmonella enterica strains, all three E. coli O157:H7 strains, four L.

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monocytogenes strains, four Shigella spp. strains, five S. aureus strains, and three C.

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jejuni strains were positive in the GeXP-PCR assay and all of the non-target

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organisms were negative in the assay. No mispriming or non-specific amplification

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was observed. None of the reactions generated more than a single peak and peaks of

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same species with different serotypes were shown in same position. In addition, the

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expected size of each pathogen amplicon was obtained only from the target

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microorganisms, resulted in 100% inclusivity and 100% exclusivity.

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3.3. The detection limits of the GeXP-PCR assay

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For each of the six pathogens, GeXP-PCR assay achieved a sensitivity of 101-102

CFU/mL for both a single bacterial target (data not shown) and when all the six

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pre-mixed bacterial targets were present (Fig. 3). The detection limit of the

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GeXP-PCR assay was 420 CFU/mL for Salmonella, 93 CFU/mL for E. coli O157:H7,

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310 CFU/mL for L. monocytogenes, 270 CFU/mL for S. aureus, 85 CFU/mL

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for Shigella spp. and 66 CFU/mL for C. jejuni (Fig. 3). This level of sensitivity is

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sufficient to allow practical detection of pathogens in a variety of samples. All six

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pathogens in the mixed culture were detected in GeXP analysis along with plasmid

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pMD19-T as positive control. They exhibited the same peak position as results from

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their respective samples with GeXP-PCR assay when performed in uniplex (Fig. 2).

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The reaction at each concentration of the template was repeated in triplicate and

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similar results were obtained each time (coefficient of variation (CV)

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dilution).

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4. Discussion

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Rapid and sensitive detection of food-borne pathogens is crucial not only to control

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and investigate food poisoning outbreaks but also to improve food safety and risk

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management. In this study, we developed and evaluated a novel application of the

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GeXP analyzer, which was capable of detecting the presence of multiple pathogens in

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a single reaction. The analytical procedure includes primer design, PCR amplification

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using both chimeric primers and universal primers, capillary electrophoretic

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separation based on the size of the PCR product followed by identification in terms of

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the expected size. Results of our analysis show that the GeXP analyzer provides a

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specific, cost-effective and high-throughput method, which allows for the rapid and sensitive detection of six food-borne pathogens.

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For the analysis of GeXP-PCR assay, we chose six of the representative bacteria

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usually associated with food-borne illnesses: Salmonella enterica, Escherichia coli

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O157:H7, Listeria monocytogenes, Staphylococcus aureus, Shigella spp., and

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Campylobacter jejuni. The GeXP method is capable of detecting these six pathogens

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in approximately 4 hr (1 hr for DNA extraction, 2 hr for PCR amplification, 45

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min/row of 8 samples for capillary electrophoretic separation, and 10 min for

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interpretation). In addition, the cost of the method for simultaneous identification of

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six food-borne pathogens is approximately $10 per test. This is, by far, cheaper than

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the $10 cost per test for each pathogen using a conventional detection method (Kim et

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al., 2007), which relies primarily on direct plating methods and biochemical tests.

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Many other studies have also reported analysis for methods of multiple microbes in

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a single sample. However, some multiplex PCR methods for the simultaneous

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detection of pathogens were developed using agarose gel analysis to detect PCR

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products, which have low sensitivity and were unsuitable for high-throughput analysis.

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In contrast, GeXP-PCR assay was a sensitive method, capable of detecting as low as

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101-102 CFU/mL of six pathogens in a single tube without any enrichment steps. The

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GeXP analyzer uses capillary electrophoretic separation of PCR products to provide

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the relative intensity versus size for all target amplicons in a DNA sample. The

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fragments corresponding to each microbe were sufficiently separated by using the

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GeXP analysis, while the conventional gel electrophoresis failed to sufficiently

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differentiate PCR products originating from the 6 pathogens (Fig. 4). Additionally, two 96-well plates can be used in parallel to analyze 192 samples in a single analysis simultaneously, indicating the high throughput of the GeXP-PCR assay.

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Each pair of target-specific primers only generated a single peak for each microbe

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species and peaks of same species with different serotypes were shown in same

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position. No non-specific or false-positive result was observed, indicating the

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specificity of the GeXP analysis. Several noise peaks were observed among the PCR

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products represented 101 CFU/mL and 102 CFU/mL in Fig. 3, however, the locations

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of the noise peaks were quite different from those of the diagnostic peaks of the

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pathogens of interest, meaning that these peaks did not interfere with the detection of

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the target pathogens.

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The GeXP multiplex PCR amplification condition was improved by using a fixed

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annealing temperature, including 2 steps with different annealing temperatures: step 1

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(first 12 cycles) was performed at a higher temperature using the gene-specific

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sequence of the chimeric primers to produce amplicons that have universal tags; step

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2 was predominantly performed using universal primers at a lower temperature in the

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subsequent cycles (numbers 13 to 32). The improved PCR condition enhanced the

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specificity and sensitivity of multiplex PCR analysis. It also minimized the occurrence

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of inferior and non-specific amplifications (data not shown). Moreover, the plasmid

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pMD-19 was added to each reaction as positive control to ascertain whether the PCR

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step and data analysis step of the assay were carried out correctly. Further evaluation

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of the GeXP-PCR assay with a larger number of real food samples from different

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sources is necessary to confirm its value and modify the analysis procedures to reduce the number of steps involved.

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In conclusion, our findings demonstrate that the GeXP multiple PCR method is a

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rapid, sensitive and high throughput method for parallel analysis of food-borne

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pathogens. It may also lead to the establishment and improvement of food safety

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systems for pathogenic bacteria.

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Funding source This work was supported by the Chongqing Science and Technology Commission

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(CSTC2011AC1056). The sponsors had no role in study design, data collection,

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analysis and interpretation, writing of the report or the decision to publish.

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Acknowledgments

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Uchenna Anunne for reviewing the manuscript.

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We are grateful to Dr. Zongyi Zou for his valuable assistance in experiment and Mr

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353

sensitive detection of 68 unique varicella zoster virus gene transcripts in five

354

multiplex reverse transcription-polymerase chain reactions. Journal of Virological

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Perry, L., Heard, P., Kane, M., Kim, H., Savikhin, S., Dominguez, W., et al. (2007). Application of multiplex polymerase chain reaction to the detection of pathogens in

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used in human medicine for Campylobacter jejuni isolated from farm animals and

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their environment in Lancashire. Journal of Antimicrobial Chemotherapy, 46(2),

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303–306. Rai, A., Kamath, R., Gerald, W., & Fleisher, M. (2009). Analytical validation of the

364

GeXP analyzer and design of a workflow for cancer-biomarker discovery using

365

multiplexed gene-expression profiling. Analytical and Bioanalytical Chemistry,

366

393(5), 1505–1511.

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363

Rodríguez-Lázaro, D., D’Agostino, M., Herrewegh, A., Pla, M., Cook, N., &

368

Ikonomopoulos, J. (2005). Real-time PCR-based methods for detection of

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Mycobacterium avium subsp. paratuberculosis in water and milk. International

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Shin, G. W., Hwang, H. S., Oh, M. H., Doh, J., & Jung, G. Y. (2010). Simultaneous quantitative

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Electrophoresis, 31(14), 2405–2410.

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pathogens using

high-resolution

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of

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374

detection

TE D

372

food-borne Salmonella. Food Control, 26(2), 369–377.

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Yang, M. J., Luo, L., Nie, K., Wang, M., Zhang, C., Li, J., et al. (2012). Genotyping of

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11 human papilloma viruses by multiplex PCR with a GeXP analyzer. Journal of

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Medical Virology, 84(6), 957–963.

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386 387

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397 398 399 400 401

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Figures Captions

406

Fig. 1. Identification of 6 food-borne pathogens. Panel (A) shows the results of

407

amplification of six food-borne pathogens in a single reaction. (B) No peak of the

408

pathogen was observed in negative control using the multiplex primer set; The red

409

peaks indicate the DNA size standard-400.

410

Fig. 2. Specificity of the GeXP-PCR assay. Panels (A-F) show the results of

411

amplification of six representative bacterial strains: (A) Listeria monocytogenes strain

412

(ATCC 15313); (B) Escherichia coli O157:H7 strain (ATCC 8739); (C) Shigella

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flexneri 2b strain (ATCC 12022); (D) Staphylococcus aureus strain (ATCC 6538); (E)

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Campylobacter jejuni strain (ATCC 33291); (F) Salmonella enterica serovar

415

Enteritidis strain (ATCC 13076). (G) Nuclease-free water was used as negative

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control, the red peaks indicate the DNA size standard-400.

417

Fig. 3. The detection limits of the GeXP-PCR assay were determined by amplifying

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10-fold diluted, purified DNA, of known concentration of each target species

419

simultaneously: (A) 4.2 × 105, 9.3 × 105, 3.1 × 105, 8.2 × 105, 8.5 × 105, 6.6 × 105

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CFU/mL of Salmonella, E. coli O157:H7, L. monocytogenes, S. aureus, Shigella spp.,

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and C. jejuni, respectively; (B) 4.2 × 104, 9.3 × 104, 3.1 × 104, 2.7 × 104, 8.5 × 104, 6.6

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× 104 CFU/mL of Salmonella, E. coli O157:H7, L. monocytogenes, S. aureus, Shigella spp., and C. jejuni, respectively; (C) 4.2 × 103, 9.3 × 103, 3.1 × 103, 2.7 × 103, 8.5 ×

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103, 6.6 × 103 CFU/mL of Salmonella, E. coli O157:H7, L. monocytogenes, S. aureus,

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Shigella spp., and C. jejuni, respectively; (D) 560, 930, 310, 270, 850, 660 CFU/mL

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of Salmonella, E. coli O157:H7, L. monocytogenes, S. aureus, Shigella spp., and C.

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jejuni, respectively; (E) 93, 85, 66 CFU/mL of E. coli O157:H7, Shigella spp., and C.

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jejuni, respectively.

429

Fig. 4. Agarose gel analysis of amplification products obtained using the multiplex

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PCR assay developed for the simultaneous detection of Listeria monocytogenes strain

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(156 bp), positive control (217 bp), Escherichia coli O157:H7 strain (242 bp),

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Shigella flexneri 2b strain (256 bp), Staphylococcus aureus strain (269 bp),

433

Campylobacter

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Enteritidis strain (320 bp). Lane M, DNA marker (DL2000; TaKaRa, Dalian, China)

435

with 2000, 1000, 750, 500, 250 and 100 bp; lane 1, Listeria monocytogenes strain

436

ATCC 15313; lane 2, positive control; lane 3, Escherichia coli O157:H7 strain ATCC

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8739; lane 4, Shigella flexneri 2b strain ATCC 12022; lane 5, Staphylococcus aureus

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strain ATCC 6538; lane 6, Campylobacter jejuni strain ATCC 33291; lane 7,

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Salmonella enterica serovar Enteritidis strain ATCC 13076; lane 8, mixture of

440

positive control and the six bacteria together.

strain

(316

bp),

and

Salmonella

enterica

serovar

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Table 1 Bacterial strains, their sources and enrichment media. Sourcea

Enrichment media

Salmonella enterica Salmonella enterica Salmonella enterica Salmonella enterica Salmonella enterica Escherichia coli Escherichia coli Escherichia coli Escherichia coli Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Listeria innocua Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Campylobacter jejuni Campylobacter jejuni Campylobacter jejuni Shigella flexneri Shigella flexneri Shigella sonnei Shigella boydii Cronobacter sakazakii Staphylococcus epidermidis Klebsiella pneumoniae Vibrio parahemolyticus Vibrio parahemolyticus Bacillus subtilis Clostridium perfringens Bacillus cereus

Enteritidis Enteritidis Typhimurium Typhimurium Choleraesuis

ATCC13076 Clinical isolate ATCC14028 Unknown ATCC10708 NCTC12900 ATCC8739 Unknown Unknown ATCC15313 Unknown Salmon Salmon Cow brain ATCC6538 CMCC26003 Meat Meat Unknown ATCC33291 Chicken sausage Clinical isolate ATCC12022 Meat ATCC25931 ATCC9207 ATCC51329 ATCC12228 ATCC700603 ATCC17802 Salmon ATCC6633 ATCC13124 ATCC11778

TSB TSB TSB TSB TSB TSB TSB TBS TSB BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BB BB BB TSB TSB TSB TSB TSB TSB TSB TSB TSB TSB TSB TSB

a

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O157：H7 O157：H7 O157：H7

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Serovar/strain

AC C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Bacteria

EP

No

Serotype 2b Serotype 2b Serotype 1

ATCC, American Type Culture Collection; NCTC, National Collection of Type Cultures; CMCC, National Center for Medical Culture Collection.

Table 2 Primers characteristics and their concentration utilized in multiplex assay. Forward primer(5’-3’) a

Reverse primer(5’-3’) a

Staphylococcus aureus Salmonella enterica Listeria monocytogenes Escherichia coli O157：H7

AGGTGACACTATAGAATAGA AACAAGAGAAGC(G/A)GTTGC AGGTGACACTATAGAATAGT GAAATTATCGCCACGTTCG AGGTGACACTATAGAATAGG ATACAACCATGAATCCGG AGGTGACACTATAGAATATG AAGGTGGAATGGTTGTCA AGGTGACACTATAGAATAATT AAC(C/A)TCTTCGCCGGACT AGGTGACACTATAGAATAAT CAGCA(A/G)GGTTTAATGGCG AGGTGACACTATAGAATATC ACGCTGTAGGTATCTCAG

GTACGACTCACTATAGGGATG TAATTGTGCCCTGTGGAA GTACGACTCACTATAGGGATC ATCGCACCGTCAAAGGAA GTACGACTCACTATAGGGAAT TTACGACGAGGTTCCACG GTACGACTCACTATAGGGATC AGC(A/G)ATTTCACGTTTTCG GTACGACTCACTATAGGGAGC AGAGACGGTATCGGAAAG GTACGACTCACTATAGGGAAC CCGCACTATCATAGCCAC GTACGACTCACTATAGGGACG CTCTGCTAATCCTGTTA

AGGTGACACTATAGAATAc

GTACGACTCACTATAGGGA

Positive control UWD-F/ UEV-Rb a

AC C

Underlined oligonucleotides are universal sequences. UWD-F/ UEV-R are the universal primers. c The forward universal primer was covalently labeled with Cy5 fluorescent dye at the 5’ end. b

Target region

Concentration (µM)

269

coa

1

320

gyrB

1

156

gyrB

1

242

rfbE

1

256

ipaH

1

316

dnaA

1

217

Random fragment

0.5

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Campylobacter jejuni

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Shigella spp.

Amplicon size (bp)

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Organism

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