Preliminary Study Of Rapid Detection Of Methicillin-Resistant Staphylococcus Aureus With Real-Time PCR Assay

Objective: To establish a duplex real-time polymerase chain reaction (PCR) assay for rapid detection of methicillin-resistant staphylococcus aureus (MRSA) and apply this method to identification of MRSA from clinical S. aureus isolates.Method: A duplex SYBR Green real-time PCR assay targeting the mecA gene and a Staphylococcus aureus (S. aureus) specific marker-nuc gene and identifying PCR products through melting curve analysis was used to rapidly identify MRSA. Agar dilution methods by using Mueller-Hinton agar containing cefoxitin (FOX) or oxacillin (OXA) were performed to give the MIC of FOX or OXA of S. aureus isolates at the same time. And then results of phenotypic methods were compared with those of genotypic methods.Results: All MRSA strains tested in the study presented mecA and nuc gene specific peaks in the melting curve analysis; methicillin-susceptible Staphylococcus aureus (MSSA) strains had only a nuc peak, methicillin-resistant Staphylococcus epidermidis (MRSE) strains had only a mecA peak, and methicillin-susceptible Staphylococcus epidermidis (MSSE) strains and strains of species other than staphylococci had no peak. MRSA strains could be detected by use of this duplex real-time PCR with an inoculum as low as 102cfu/mL. Among 131 S. aureus isolates tested, 29.01%(38/131) were positive for mecA gene and all were positive for nuc gene in the duplex real-time PCR.The agreement between simplex and duplex real-time PCR was 100% for positive results. Positive rate of FOX susceptibility test was 30.53%(40/131) in those 131 S. aureus isolates, including 40 resistant, 88 susceptible and 3 intermediate strains, from which OXA susceptibility testing distinguished 34.35%(45/131) resistant and 86 susceptible strains. Taking genotypic method as gold standard, agar dilution method of FOX has a 94.7% sensitivity, a 93.5% specificity, a positive predictive value of 90.0% and a positive likelihood ratio of 14.57, while that of OXA has a 92.1% sensitivity, a 89.2% specificity, a positive predictive value of 77.8% and a positive likelihood ratio of 8.53.Conclusion: This study shows that the duplex real-time PCR assay with primers directed to mecA and nuc genes is a valuable tool for rapid and precise identification of MRSA and methicillin-resistant coagulase-negative staphylococci (MR-CoNS).