单管高灵敏度等温扩增技术快速检测甲型H1N1流感病毒  

Sensitive Detection of Influenza A H1N1 Virus by Isothermal Amplification in A Single Tube

作　　者：成思佳[1] 陈之遥 初亚男[1] 崔仑标[3] 史智扬[3] 马寅姣[1] 周国华[1,2] 

机构地区：[1]中国药科大学生命科学与技术学院,南京210009 [2]华东医学生物技术研究所,南京210002 [3]江苏省疾病预防控制中心微生物检验科,南京210009

出　　处：《分析化学》2011年第3期335-340,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(面上项目)(No.20975113);江苏省科技支撑计划(社会发展)(No.BE2008623);江苏省基础研究计划(自然科学基金)(No.BK2008067);中央高校基本科研业务费专项资金青年项目(No.JKQ2009027)资助

摘　　要：建立了单管逆转录环介导等温扩增法(RT-LAMP)快速检测甲型H1N1流感病毒的方法。针对甲型H1N1流感病毒的M基因和HA基因的保守区,设计了两组特异性引物,分别用于筛选甲型流感病毒及鉴定甲型H1N1流感病毒。对反应体系中的关键因素进行优化,反应结果可直接通过浊度或者SYBR GreenⅠ荧光进行判定。本方法最低可检测到10拷贝/管的甲型H1N1流感病毒。为验证方法的可行性,对30例临床样本进行了检测,与美国疾病控制与预防中心(CDC)的TaqMan方法进行对比,检测的灵敏度和特异性分别为92%和100%。本方法实现了单管快速检测甲型H1N1流感病毒,为甲型H1N1流感病毒的现场检测提供了新方法。A single-tube reverse transcription loop-mediated isothermal amplification（RT-LAMP） assay was developed for the detection of influenza A H1N1 virus.First,two sets of primers were designed based on the conserved regions of both the M gene and the HA gene for the screening of influenza A virus and the identification of influenza A H1N1 subtype.Optimization of RT-LAMP reaction at various conditions was then carried out by using the above primers.Positive reactions can be readily observed by a visual inspection based on the turbidity or SYBR Green Ⅰ fluorescence.The detection limit of this method was 10 RNA copies per reaction,higher than that of the Centers for Disease Control and Prevention TaqMan assay.To investigate the feasibility of the RT-LAMP assay,30 clinical specimens were tested,and the sensitivity and specificity were 92% and 100%,respec-tively,in comparison with the Centers for Disease Control and Prevention TaqMan assay.Therefore,the RT-LAMP assay proposed here is a perspective tool for the rapid screening of influenza A H1N1 virus at resource-limited areas.

关 键 词：甲型H1N1流感病毒 逆转录环介导等温扩增 SYBR Green Ⅰ 

分 类 号：R440[医药卫生—诊断学]