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

Abstract

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 TaqMan® probes were designed to target these three pathogens. Multiplex real-time PCR was carried out with TaqMan® 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 109 CFU/ml for E. coli O157:H7, 103 to 109 CFU/ml for Salmonella and 101 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, 103 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. TaqMan® 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.