A NOVEL MULTIPLEX PCR SYSTEM FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B, TSST, NUC AND FEM GENES OF STAPHYLOCOCCUS AUREUS IN FOOD SYSTEM

Staphylococcal enterotoxin B (SEB), toxic shock syndrome, the superantigens are responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. An easy and quick system is desirable to detect toxin-producing strains. In this report, we described standardization of a novel multiplex-polymerase chain reaction (PCR) assay for simultaneous detection of four important genes associated with the Staphylococcus aureus viz., SEB, Tsst, genus-specific nuclease (nuc) and Fem genes along with an internal amplification control (IAC), which has now become mandatory in diagnostic PCRs, particularly when tested on environmental or food samples. This mPCR method is sensitive enough to detect cells as low as 103 cfu/mL or /g of the food samples. When evaluated on 136 food and environmental samples, the system detected 4 SEB-positive S. aureus strains. The S. aureus strains that have been identified to contain the SEB gene in the mPCR were unequivocally detected for the toxin expression by the TECRA kit. mPCR produced a 100% correlation with conventional identification method. As SEB and Tsst are qualified as biowarfare molecules, this system is of immense help in detecting them during emergencies of biological war and suspected outbreaks of S. aureus food poisoning directly from the food and environmental samples. PRACTICAL APPLICATIONS The virulence-associated genes targeted in this study are super antigenic in nature and are responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome, and these molecules are also qualified as biowarfare agents. The high throughput and cost-effective multiplex PCR method reported here could identify all these genes successfully from both artificially spiked and natural food samples and may also find its application in detection of these toxin-producing Staphylococcus aureus from clinical and environmental samples.