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.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
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
