Optimization and evaluation of a multiplex PCR assay for detection of Staphylococcus aureus and its major virulence genes for assessing food safety

Staphylococcus aureus is a natural commensal microflora of humans which causes opportunistic infections due to its large arsenal of exotoxins, invasion, immune evasion, and antibiotic resistance mechanisms. The primary goal of this study is to develop a multiplex PCR (mPCR) assay for simultaneous detection of Staphylococcus aureus (nuc) and its virulence genes coding for prominent exotoxins namely alpha hemolysin (hla), enterotoxins A (sea), enterotoxin B (seb), toxic shock syndrome toxin (tsst-1), and the gene coding for methicillin resistance (mecA). A competitive internal amplification control (IAC) was included in the assay to exclude the false negative outcomes. Highly specific primer pairs were designed for the target genes using in silico resources. At the outset, monoplex PCRs were standardized using reference S. aureus strains. Primer specificity to the target genes was authenticated through restriction digestion analysis of amplified PCR products. Multiplex PCR was optimized in increments of one gene starting with nuc and IAC amplified simultaneously using one pair of primers (nuc) in a competitive manner. The mPCR assay was found to be highly sensitive with a detection limit of similar to 10 CFUs per reaction for pure cultures. Multiplex PCR assay was further evaluated on the retail and processed food samples to test the prevalence of S. aureus and study their exotoxin profiles. Of the 57 samples examined, 13 samples (22.80%) were found to be contaminated with S. aureus whose DNA was extracted after a 6-h enrichment period. Among these, a high percentage of hemolytic and enterotoxin A positive strains were encountered. The mPCR assay developed in this study would be a useful tool for rapid and reliable monitoring of S. aureus for food quality testing and from clinical infections.