Simultaneous detection of Escherichia coli O157:H7, toxigenic Vibrio cholerae, and Salmonella typhimurium by multiplex PCR

Background: Escherichia coli O157:H7, Vibrio cholerae, and Salmonella typhimurium are pathogenic bacteria found in contaminated water and food. No assay method is currently available on simultaneous detection or identification of all the three pathogens. Our aim was to develop a rapid and reliable method for this purpose. Materials and methods: A protocol for sample collection, and a PCR procedure was designed specifically for the assay. Selected fragments of 239 bp, 432 bp, and 360 bp for E. coli O157 lipopolysaccharide (LPS) gene (rfbE), V. cholerae toxin gene (ctx), and Salmonella typhimurium putative cytoplasmic protein gene (STM4497), respectively, were amplified from the extracted bacterial DNA samples in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. Results: All unknown samples were verifiably identified. The assay was sensitive enough to detect and identify as few as 100 cells of E. coli O157:H7, V. cholerae and Salmonella typhimurium. The presence of other bacteria did not interfere with the analysis. Conclusion: This assay is a specific and reliable tool which allows cost-effective detection of all three bacterial pathogens in one reaction tube.