Abstract: Objective:To develop a duplex quantitative real-time PCR assay for rapid detection of Staphylococcus aureus and identification of methicilin resistant Staphylococcus aureus(MRSA). Methods:Two pairs of specific primers and TaqMan fluorescent probe were designed according to the conserved region of NUC gene sequences of Staphylococcus aureus and MecA gene sequences of MRSA,respectively. The 5' end labeled with different fluorescent reporter groups (FAM and HEX) and the 3' end labeled with fluorescence quenching group were used to detect Staphylococcus aureus and identify the gene of MRSA in order of evaluate its sensitivity and specificity. Results:The genes of NUC and MecA were specifically amplified in the duplex PCR reaction respectively. The MRSA in the study presented MecA and NUC gene,while methicillin susceptible Staphylococcus aureus strains had only a NUC gene; the same species of Staphylococcus epidermidis,Streptococcus faecalis and Micrococcus luteus had no specific amplification gene; at least 100MRSA strains were detected by using this duplex real-time PCR. Conclusions:The duplex real-time PCR assay can not only monitor the community and clinical-acquired MRSA infection, but provide necessary basis for pharmacotherapy to MRSA infection patients.