Establishment of a Duplex Fluorescent Quantitative PCR Assay for Staphylococcus aureus Detection Based on Pangenome Analysis

(1) Background: Staphylococcus aureus is a common human respiratory pathogen. Its widespread antimicrobial resistance often leads to severe infections. However, the high genomic diversity among strains frequently causes false negatives in conventional PCR assays. (2) Methods: To address this limitation, we developed a highly sensitive and specific duplex fluorescent quantitative PCR (qPCR) method for S. aureus detection based on pangenome analysis. Conserved core genome sequences of S. aureus were identified using a pangenomic approach. Two highly conserved and species-specific genes—isdA and recQ—were selected as target loci for the duplex qPCR assay. A TaqMan probe-based duplex fluorescent qPCR assay was established, enabling simultaneous detection of both isdA and recQ genes in a single reaction. (3) Results: The assay demonstrated detection sensitivities of 1.335 × 103 copies/μL for isdA and 7.265 × 102 copies/μL for recQ. The intra-assay and inter-assay coefficients of variation (CV) for Ct values were below 1.86% and 2.87%, respectively, indicating excellent reproducibility. Specificity analysis revealed no cross-reactivity with genomic DNA from 14 common respiratory pathogens, including Pseudomonas aeruginosa and Streptococcus pneumoniae. (4) Conclusions: Due to its high specificity and sensitivity, this duplex qPCR assay effectively overcomes limitations of single-target PCR methods, such as missed detections and prolonged turnaround times, and holds significant potential for practical diagnostic applications.