Simultaneous Detection of Food Pathogens, Staphylococcus aureus, Salmonella Enterica, Bacillus cereus and Vibrio parahaemolyticus by Multiplex Real-Time Polymerase Chain Reaction

The study established a multiplex real-time polymerase chain reaction (PCR) for simultaneous detection and quantitation of Staphylococcus aureus, Salmonella Enterica, Bacillus cereus and Vibrio parahaemolyticus in the same reaction at one time, thus reducing the amounts of reagents and cutting down on the labor and time. Four sets of genetic marker specific primers corresponding to SAOUHSC_02297, invA, hbl and tlhA gene, and probes were labeled with FAM, HEX, TEXAS RED and Cy5 respectively. Multiplex real-time PCR was carried out with an iQTM Multiplex Powermix Kit (BIO-RAD) Sequence Detection System. The result showed that the correlation coefficient between multiplex real-time PCR estimates and plate counts of 10-serial dilutions in specific bacteria was above 0.98, independent of 101-4-fold numbers of three other pathogenic strains. With optimized conditions, the lowest detection concentrations of four pathogens were 102.5 CFU/mL for Sta. aureus, S. Enterica and V. parahaemolyticus and 103.5 CFU/mL for B. cereus. The duration of the entire experiment from DNA isolation and purification to PCR amplification was less than 12 h.