Simultaneous quantitation of Escherichia coli O157:H7, Salmonella and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation

The objectives of this study were to establish a real-time multiplex polymerase chain reaction (PCR) for simultaneous quantitation of Escherichia coli O157:H7, Salmonella and Shigella that have been implicated in a number of foodborne disease outbreaks. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqManRTM probes were designed to target these three pathogens. Multiplex real-time PCR was carried out with TaqManRTM Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated by plotting the threshold cycle (Ct) value against log10 CFU/ml by linear regression to analyze the results for each pathogen. With optimized conditions, the quantitative detection ranges of the real-time multiplex PCR for pure cultures were 102 to 10 9 CFU/ml for E. coli O157:H7, 103 to 109 CFU/ml for Salmonella and 10 1 to 108 CFU/ml for Shigella. When this established multiplex real-time PCR system was applied to ground beef samples, the lowest detection concentration of three pathogens were increased to 105 CFU/g for E. coli O157:H7, 10 3 CFU/g for Salmonella and 104 CFU/g for Shigella. Immunomagnetic separation was then used to isolate E. coli O157:H7 and Salmonella from the beef samples. The lowest detection concentrations of three pathogens were reduced to 103 CFU/g. TaqManRTM real-time PCR, combined with IMS has the potential to be a faster and more reliable method for rapid quantitation of E. coli O157:H7, Salmonella and Shigella in food, which will take 3 h for the whole process.