双重实时荧光PCR快速检测耐甲氧西林金黄色葡萄球菌方法的建立

Research on rapid detection of methicillin resistant Staphylococcus aureus by duplex realtime-PCR

目的:建立一种能快速检测金黄色葡萄球菌,同时能快速鉴别是否具有耐甲氧西林基因的双重实时荧光PCR方法。方法:以金黄色葡萄球菌特异NUC基因的保守区为靶区域设计特异引物和TaqMan荧光探针,同时针对耐甲氧西林特异MecA基因设计另一对引物和探针;2条探针的5'端标记不同荧光报告基团(FAM和HEX),3'端标记荧光淬灭基团,建立双重实时荧光PCR方法,检测样本中的金黄色葡萄球菌以及鉴别其是否具有耐甲氧西林基因,并对双重实时荧光PCR方法的的特异性和灵敏度进行评价。结果:双重荧光PCR方法可对金黄色葡萄球菌及耐甲氧西林基因分别进行特异扩增,两者均为阳性时可判为耐甲氧西林金黄色葡萄球菌;金黄色葡萄球菌特异基因特异扩增阳性而耐甲氧西林基因特异扩增阴性可判为甲氧西林敏感的金黄色葡萄球菌;对同种属的表皮葡萄球菌、粪链球菌、藤黄微球菌等病原体均无扩增;双重实时荧光PCR方法的灵敏度最低可检测至100个菌体。结论:建立的实时荧光PCR检测耐甲氧西林金黄色葡萄球菌的方法不仅能实现对耐甲氧西林金黄色葡萄球菌社区感染和临床感染的监控,同时也可为临床上耐甲氧西林葡萄球菌感染患者的用药提供必要的依据。

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.