耐甲氧西林金黄色葡萄球菌mecA基因检测的LAMP方法建立  

Establishment of loop-mediated isothermal amplification technique for detection of mecA gene of methicillin-resistant Staphylococcus aureus

作　　者：陈凤华 欧红玲 吴丽芬[2] 王岩 白静 张巧云 马盈盈 王欣茹 

机构地区：[1]第二炮兵总医院医学检验科,北京100088 [2]天津医科大学总医院风湿免疫科

出　　处：《西北国防医学杂志》2015年第10期657-660,共4页Medical Journal of National Defending Forces in Northwest China

基　　金：全军"十二五"面上课题资助项目(CWS11J205);军队"十二五"重大专项子课题资助项目(424135S)

摘　　要：目的:探讨环介导等温扩增技术(LAMP)检测耐甲氧西林金黄色葡萄球菌(MRSA)mecA特异性基因的可行性和可靠性。方法:建立LAMP反应体系,优化反应条件,进行灵敏度和特异性试验。结果:LAMP反应检测mecA基因的最佳引物组为引物组2+环引物1,最佳反应温度为62℃,LAMP检测mecA基因的最低限为14.7pg/μl,PCR检测mecA基因的最低限为147pg/μl(引物量与LAMP一致),敏感性较PCR高10倍。LAMP检测mecA基因的特异度为100%,假阳性率为15%,假阴性率为0。结论:LAMP是一种操作简单、检测灵敏度和特异度高的扩增方法,仅靠检测mecA基因来鉴别MRSA假阳性率高,需结合金黄色葡萄球菌特异nuc基因同时扩增鉴定效果最好。Objective：To explore the feasibility and reliability of loop-mediated isothermal amplification（LAMP）for rapid detection of methicillin-resistant Staphylococcus aureus specific gene mecA.Methods：LAMP reaction system was established,the reaction conditions was optimized,and the sensitivity and specificity were tested.Results：The optimal primer set for mecA gene in LAMP reaction was primer 2plus loopprimer1.The optimal reaction temperature was 62℃.The minimal detection limit of mecA gene for LAMP was 14.7pg/μl,while it was 147pg/μl for PCR using same amount of primers with LAMP,indicating 10 times higher sensitivity in LAMP than that in PCR.The specificity of LAMP was 100%,the false positive rate was 15% and the false negative rate was 0%.Conclusion：LAMP is easy to operate and has high sensitivity and specificity.However,this method should be combined with gene nuc amplification to decrease the false positive rate for MRSA detection.

关 键 词：实验室诊断 环介导等温扩增技术 耐甲氧西林金黄色葡萄球菌 快速检测 

分 类 号：R446.5[医药卫生—诊断学]