Rapid and Simultaneous Detection of Five Classical Staphylococcus aureus Enterotoxin Genes via Multiplex Melting Curve Analysis Using Self-Quenching Primers

ng/μL, demonstrating 100% analytical specificity with no cross-reactivity against a panel of 12 non-target bacterial strains. Verification using 171 S. aureus strains isolated from milk samples revealed an overall enterotoxin prevalence of 47.95%, with SEA (19.30%) and SEE (12.28%) being the most predominant genotypes. Compared to conventional PCR, the proposed method demonstrated 100% specificity and 97.62% sensitivity, offering a robust, cost-effective, and high-throughput tool for the routine surveillance of enterotoxigenic S. aureus in the dairy industry.