2.1 Bacterial strainsThe bacterial strains were obtained from the Pasteur Institute, Tehran, Iran and used in this study (Table 1). Clinical isolates of the three most important foodborne bacterial pathogens including Salmonella and Shigella were obtained from patients admitted to Children's Medical Center and Baqiyatallah Hospitals in Tehran, Iran, during 2012–2014. Subsequently, identification of the references and clinical strains was confirmed by culture, biochemical testing by the API test system (BioMérieux, Marcy-l'Étoile, France), and slide agglutination with serovar specific antisera (Staten Serum Institute, Copenhagen, Denmark). V. cholerae isolates were provided by the Molecular Biology Research Center affiliated to Baqiyatallah Hospital.All bacterial strains were grown either on Brain Heart Infusion (BHI; Difco Laboratories, Detroit, MI, USA) or Luria–Bertani (LB) broth (Merck, Darmstadt, Germany) at 37°C for 18–24 hours.
2.2 DNA extractionGenomic DNAs from all microorganisms were extracted using the DNA extraction kit (DNP, DNA Extraction Kit; Cinagene Company, Tehran, Iran) according to the manufacturer’s instructions. DNA concentration and purity were spectrophotometrically assessed by reading A260 and A280 and confirmed by visualization on 1% agarose gel. Then, DNA was diluted to 1 mg/mL in nuclease-free water and stored at –20°C until required for analysis.
2.3 Primers and multiplex PCR conditionsThe AlleleID software version 7.01 (Premier Biosoft Int., Palo Alto, CA, USA) was used for all oligonucleotide primers designed in this study. All primers were purchased from Bioneer (Daejeon, South Korea). The in silico specificity was analyzed using the Basic Local Alignment Search Tool (BLAST) from the GenBank database. The characteristics of the primers used for multiplex PCR are given in Table 2.PCR was carried out with a 50-μL mixture containing 10mM Tris–HCl (pH 8.3), 50mM.In this study, we used KCl, 1.5mM MgCl2, 1 U of Taq DNA polymerase (Promega, Madison, WI, USA), 0.2mM deoxynucleoside triphosphate, a 0.1μM concentration of each primers, and 5 μL of the DNA sample.Multiplex PCR was performed under the following conditions: 35 cycles with heat denaturation at 95°C for 30 seconds, primer annealing at 60°C for 30 seconds, and DNA extension at 72°C for 60 seconds in Eppendorf gradient master cycler (Roche, Mannheim, Germany). The amplified DNA was separated by 1% agarose gel electrophoresis, stained with ethidium bromide, and visualized by UV transilluminator.
2.4 Sensitivity and specificityTo determine the sensitivity of the multiplex PCR assay, 10-fold serial dilutions were made from extracted genomic DNA (498 ng/μL), and the detection limit of the multiplex PCR was defined as the lowest concentration of DNA that could be amplified. The specificity of multiplex PCR was evaluated using three species including Staphylococcus aureus PTCC (Persian Type Culture Collection) 1189, E. coli ATCC (American Type Culture Collection) 25922, and Citrobacter freundii ATCC 8090 as negative controls.
ResultsThe multiplex PCR using three sets of primer pairs targeted for the invA, putative integrase, and ompW genes, correctly identified Salmonella spp., Shigella spp., and V. cholerae and differentiated them by the different-size bands products: three positive bands, which consist of invA (403 bp), putative integrase (159 bp), and ompW (592 bp) PCR products (Figure 1). No amplification products were observed in multiplex PCR with any of the other microorganisms subjected to the assay (Table 1). The sensitivity of the multiplex PCR was assessed to be 5 ng/μL of the pure DNA.
DiscussionSalmonella spp., Shigella spp., and V. cholerae are responsible for large numbers of intestinal infections in humans worldwide. Molecular techniques, such as multiplex PCR, are proving useful in detection of pathogens in a wide spectrum of matrices 10, 19. This technique enables us to identify these three pathogens at one experiment, obviating the need for three separate experiments. The use of multiplex PCR substantially reduces the time and manpower required when compared with conventional methodologies. Here, we report a multiplex PCR assay for detection of Salmonella, Shigella, and V. cholerae based on invA, ompW, and putative integrase genes, respectively. Previous studies indicated that these genes are conserved in each species. Many studies noted that invA is a specific and sensitive target for detection of Salmonella spp. 20, 21. Also, the ompW gene has been previously used for identification of V. cholerae, owing to its specificity . Furthermore, restriction fragment length polymorphism analysis and nucleotide sequence data have shown that the ompW gene is highly conserved among all V. cholerae biotypes, suggesting the ompW gene can be considered a good target for the specific identification of V. cholerae strains .Unlike the above-mentioned species, Shigella genomes have a high level of similarity with the E. coli genome; hence, the whole sequences of Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei have ∼3 Mb of genomic DNA in common with all sequenced E. coli genomes . However, based on the comparative genomic analysis, a specific target known as putative integrase locus, conserved in all Shigella species, was subjected to identification of Shigella species. Hence, Shigella-specific primers were designed based on putative integrase locus. The results also showed that this locus is a suitable target for specific identification of Shigella species.In many research studies, multiplex PCR has been applied for rapid identification of diarrheal agents 25, 26. All of these studies noted that multiplex PCR is a reliable, useful, and cost-effective assay, which is consistent with our results. Jin et al  studied foodborne pathogenic bacteria including C. jejuni, Shigella, Salmonella, Vibrio parahaemolyticus, S. aureus, E. coli O157:H7, and several other bacterial species and showed that multiplex PCR is time-saving assay in comparison with conventional PCR. Furthermore, Paniagua et al , who described the detection of different foodborne pathogens by multiplex PCR, noted that this method could be useful for quick detection of foodborne pathogens.There are inconsistent reports about the sensitivity of multiplex PCR. According to Tsai et al , the sensitivity of multiplex PCR is considerably lower than that of monoplex PCR because of the primers’ interference, so that it can be decreased several times compared with conventional PCR. However, Al-Talib et al  showed that multiplex PCR has a high level of sensitivity, and it might be useful as an alternative diagnostic tool for diarrheal diseases. In our study, a high level of sensitivity (5 ng/μL) was also observed. It appears that the sensitivity of multiplex PCR is related to primer length and can be enhanced by shortening the primers’ length. However, this modification leads to low specificity.The infections caused by enteric pathogens comprise second commonest medical problems after respiratory infectious disease 31, 32. Salmonella, Shigella, and Vibrio are among the most prevalent and endemic food and water- borne pathogens in Iran 33, 34, 35, 36. Rapid and simultaneous detection of these common bacteria in is extremely important to ensure food and water safety. For this purpose, we developed and successfully applied a multiplex PCR for the rapid identification of Salmonella spp., Shigella spp., and V. cholera. This technique decreases the test time of PCR. This method is simple and rapid, and the results obtained proved to be highly specific and sensitive and can be expanded to additional species. Moreover, multiplex PCR may provide an epidemiological tool to investigate the wide spread of diarrheagenic pathogens in various areas worldwide.