A multiplex RTi-PCR reaction for simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus on fresh, minimally processed vegetables

In this work, a new multiplex single-tube real-time PCR approach is presented for the detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus, three of the more frequent food-borne bacterial pathogens that are usually investigated in a variety of food matrices. The study includes the design and specificity testing, of a new primer and probe specific for Salmonella spp. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the β-glucuronidase (uidA, E. coli) and Thermonulease (nuc, Sta. aureus) genes, and in the replication origin sequence (oriC, Salmonella spp.). Melting-curve analysis using a SYBR Green I RTi-PCR approach showed characteristic Tm values demonstrating the specific and efficient amplification of the three fragments. Subsequently, a TaqMan RTi-PCR approach was settled, using FAM, NED and VIC fluorescently labelled specific probes for an automated detection. It was equally sensitive than uniplex RTi-PCR reactions in Sta. aureus and E. coli O157:H7, using same amounts of purified DNA, and allowed detection of 10 genome equivalents in the presence of 102 or 104 genome equivalents of the other two pathogens. Finally, it was tested in artificially inoculated fresh, minimally processed vegetables, revealing a sensitivity of 103 CFU g−1 each of these pathogens in direct detection, following DNA extraction with DNeasy Tissue Kit (Qiagen). The multiplex RTi-PCR developed scored the sensitivity recognised for PCR in food and it allows a high-throughput and automation, thus it is promising as a rapid and cost-effective test for the food industry.