Objective To establish the method of identifying MRSA with Taqman-fluorescence quantitative PCR basing on mecA/nuc/fem B three gene combined detecting.Methods Taking the coagulase positive MRSA,which isolated from the clinical samples and confirmed by VITEK 2 compact microbial analyzer,as the research obj ect,designed mecA/nuc/fem B specific PCR primers and Taqman fluorescent probe by bio-software PrimerPremier 5 and Designer Beacon 7,FAM,HEX and ROX markers were used to label the fluorescent probe at 5’,and the end of 3’was labeled with BHQ1,detected by fluo-rescence quantitative PCR instrment.Results ①1 g/dl gel electrophoresis results showed that the primer’s specificity of mec A/nuc/fem B were good,and molecular weight of the amplification band consistent with the expected molecular weight and no non-specific amplification band.②Three genes were obtained specific amplification in a single tube single channel and single tube multiple channel detection in PCR,and the three gene amplification effect in a single tube single tube single chan-nel and multichannel PCR similar.Conclusion Successfully established a method of multi channel Taqman-probe fluores-cence quantitative PCR identification of MRSA,mec A/nuc/fem B combined detection can effectively differentiate coagulase negative and positive MRSA,improve the accuracy of identification.%目的建立基于mec A／nuc／fem B三基因联合的 Taqman-探针荧光定量 PCR鉴定耐甲氧西林金黄色葡萄球菌（MRSA）的方法。方法以常规检验标本中分离和采用 VITEK 2 Compact微生物分析仪鉴定为凝固酶阳性的 MRSA为研究对象，通过PrimerPremier5.0和Beacon Designer 7软件设计针对mec A／nuc／fem B特异性PCR引物及Taqman荧光探针，荧光探针5’端分别采用 FAM，HEX及 ROX标记，3’端采用BHQ1标记，在荧光定量PCR仪进行检测。结果①1 g／dl凝胶电泳结果显示mec A／nuc／fem B三个基因引物特异性较好，扩增出的条带分子量与预期分子量一致且未见非特异性扩增；②在单管单通道及单管多通道的 PCR检测中 mec A／nuc／fem B均获得特异性扩增，且三个基因在单管多通道的PCR扩增效果与单管单通道的相类似。结论成功建立了多通道 Taqman-探针荧光定量 PCR鉴定 MRSA的方法， mec A／nuc／fem B三种基因联合检测可有效区分凝固酶阴性和阳性的 MRSA，提高鉴定 MRSA的准确率。
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