基于TaqMan探针三重荧光PCR检测MRSA  

Detection of MRSA by TaqMan probe- based triple fluorescence PCR

作　　者：袁慕云[1] 张旺[1] 卓锦雪[1] 谢力[1] 陈碧玲[1] 许龙岩[1] 

机构地区：[1]广东出入境检验检疫局检验检疫技术中心,广东广州510623

出　　处：《中国卫生检验杂志》2014年第2期239-241,252,共4页Chinese Journal of Health Laboratory Technology

基　　金：广东出入境检验检疫局科技计划项目(2012GDK35)

摘　　要：目的建立基于TaqMan探针三重荧光PCR检测耐甲氧西林金黄色葡萄球菌(MRSA)方法。方法根据nuc(GenBank:EF529607.1)、mecA(GenBank:JN108029.1)和femA gene(GenBank:DQ352463.1)基因序列,分别设计特异性引物和Taqman探针,nuc探针5、端标记TET、mecA探针5、端标记HEX、femA探针5、端标记FAM,建立基于Taqman探针三重荧光PCR检测MRSA的方法,并对69株金黄色葡萄球菌分离株进行基因检测与耐药表型比较。结果 MRSA出现femA、mecA、nuc基因扩增曲线,而表皮葡萄球菌等14株对照菌株扩增结果呈阴性;femA、mecA、nuc基因的扩增效率分别为104%、90.3%、98%,线性系数R2分别为0.999、0.994、0.998;69株分离株中femA、nuc基因阳性率为100%,mecA基因阳性率为13.04%,mecA与耐甲氧西林表型符合率为100%。结论建立的方法特异性强、准确,为金黄色葡萄球菌鉴定和耐药基因分析提供参考依据。Objective To establish a method for detection of MRSA by Taqman probe - based triple real time PCR. Methods PCR primers and Taqman probes were designed based on the gene sequence of nuc （GenBank： EF529607.1 ） , mecA （GenBank： JN108029.1 ） and fermi gene （GenBank： DQ352463.1 ）, labeling TET at nuc probe 5 ＂terminal, mecA with HEX, femA with FAM. Taqman probe based triple real time PCR was established for detection of MRSA, and 69 staphylococcus aureus isolates were carried out gene detection and resistance phenotype comparison. Results Amplification offemA, mecA and nuc were posi- tive in MRSA, while the other 14 reference isolates were negative. The amplifying effieiencies were 104%, 90.3%, 98% in turn, with the correlation coefficients of 0.999, 0. 994, 0.998. The positive rates offemA, nuc gene were both 100% in 69 iso- lates, while that of meeA was 13.04%. The coincidence rate of mecA gene and MRSA was 100%. Conclusion The estab- lished method is accurate and highly specific, providing references for staphylococcus aureus identification and drug - resistance gene analysis.

关 键 词：MRSA TAQMAN探针 三重荧光PCR 耐药基因 检测 

分 类 号：R378.11[医药卫生—病原生物学]