AimsTo develop a multiplex PCR assay coupled with selective enrichment step to detect major virulence-associated genes of enterotoxigenic Stap-hylococcus aureus and evaluate the same directly on contaminated food samples. Methods and ResultsThe most important virulence-associated genes of Staph.aureus, which are commonly related to food safety issues, are targeted in this study. They include five major enterotoxigenic genes-sea, seb, sec, seg and sei, tst-which encodes TSST-1, mecA-which confer methicillin resistance and coa-for the enzyme coagulase along with an internal amplification control (IAC) to rule out false-negative result. A modified mannitol salt broth (MSB) supplemented with sodium pyruvate was used for selective enrichment of Staph.aureus from food samples prior to PCR. Evaluation of efficiency of different media revealed that enrichment of samples in modified MSB followed by PCR resulted in specific, sensitive and effective amplification of the targeted genes in comparison with other enrichment media. Incorporation of bovine serum albumin (BSA) as PCR enhancer improved the intensity of amplicons. The standardized multiplex PCR (mPCR) format was able to detect all the target genes at a bacterial load of 10(6)CFUml(-1)in any sample. The PCR results were unequivocally correlated with the conventional methods when the mPCR format was assessed on a total of 91 Staph.aureus isolates. The entire assay was found to be effectual when evaluated on naturally contaminated food samples. ConclusionsThe combinatorial approach involving selective enrichment followed by mPCR developed in this study was found to be effective for the detection of toxigenic Staph.aureus directly from various food sources. Significance and Impact of the StudyThe developed format would find a promising application in early detection of food contaminations as well as in the diagnosis of food poisoning due to Staph.aureus.