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

• Self-quenching primers enable cost-effective, sensitive PCR. • Assay achieves 10 -2 ng/μL LOD with 100% specificity. • SEA and SEE genotypes predominate in milk-isolated S. aureus. • Rapid, high-throughput workflow completes within 3 hours. Staphylococcus aureus is a ubiquitous foodborne pathogen whose pathogenicity is primarily driven by staphylococcal enterotoxins (SEs), necessitating rapid and reliable screening methods to prevent disease outbreaks. Current detection techniques, however, are often limited by high costs, post-amplification contamination risks, or labor-intensive protocols. To address these challenges, this study developed a novel, closed-tube multiplex melting curve assay utilizing self-quenching primers for the simultaneous detection of the five classical enterotoxin genes. The assay successfully differentiated all five target genes within a single reaction based on distinct melting temperatures and achieved a limit of detection of 10 -2 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.