ObjectiveTo establish a quantitative realtime polymerase chain reaction（qPCR） assay for rapid detection of Staphylococcus aureus (SA) and its mecA gene, rapidly and accurately diagnose SA infection as well as preliminarily determine its drug resistance.MethodsSequences of nuc, atl, icaB, fnbA, hla, and srap genes of SA were downloaded from NCBI database for screening of markers, and sequence of mecA gene was used for screening of methicillinresistant SA (MRSA); sequence alignment was conducted by DNA MAN, conserved region of each gene sequence was employed for designing primers and fluorogenic probes. Simplex and duplex qPCR were established, and gene sequences with the best detection performance were screened by clinical and standard strains as markers, a duplex qPCR system for identifying SA and drug resistance was developed and evaluated.ResultsThe atl gene（CP009361.1：1010217-1010341）and mecA gene （KF058908.1：1715-1843）possessed the best detection performance, and were used as detection markers in the duplex qPCR system. qPCR system could amplify both targets in a range of 2.0×1028 copies/mL with a strong linear relationship and lower detection limits for both targets reached 4 copies/PCR. Among 335 positive culture specimens for SA (including 94 MRSA) from patients, 335 and 94 specimens were detected SA and MRSA by duplex qPCR respectively; among 95 negative culture specimens for SA from patients, 17 and 4 specimens were detected SA and MRSA by duplex qPCR respectively, PCR products were sequenced, the homology with standard strain were all≥90%. Time from specimen processing to result reporting was≤2.0 hours by duplex qPCR method.ConclusionqPCR method is simple, rapid, sensitive, and specific, it’s a promising way to improve the diagnostic efficacy of SA infection and achieve the rapid detection, which contributes to the early precision treatmen