H7N9人禽流感病毒RT-LAMP快速检测方法的建立  

Establishment of a rapid method for detection of H7N9 human avian influenza virus by a reverse transcriptase loop-mediated isothermal amplification

作　　者：崔淑娟[1] 严燕[2] 任海林[1] 彭晓旻[1] 杨鹏[1] 王全意[1] 

机构地区：[1]北京市疾病预防控制中心传染病地方病控制所,北京100013 [2]国家林业局幼儿园,北京100013

出　　处：《中国卫生检验杂志》2015年第15期2456-2458,2462,共4页Chinese Journal of Health Laboratory Technology

基　　金：北京市科技计划课题(D141100003114002);首都卫生发展科研专项项目(首发2014-1-1011);北京市卫生系统高层次卫生技术人才培养计划项目(2013-3-098)

摘　　要：目的采用RT-LAMP技术建立一种针对H7N9人禽流感病毒的特异、灵敏、快速、可视、简便的检测方法。方法根据H7N9人禽流感病毒HA基因的保守序列,利用Primer Explorer V4软件,设计一套针对HA基因的7个区域的5条特异性引物,优化反应体系与扩增条件,并验证其特异度与灵敏度。结果该方法能检测出H7N9亚型流感病毒,而对H1亚型、H3亚型、H5亚型、H6亚型、H9亚型流感病毒、乙型流感病毒、人副流感病毒、呼吸道合胞病毒、人冠状病毒、人偏肺病毒、人腺病毒、人博卡病毒均无扩增反应,其最低检出限为0.01 pg。另外,扩增反应只需要45 min即可判读结果,并可通过反应液是否有沉淀或向反应液中加入荧光染料来对结果进行可视化诊断。结论本研究建立的RT-LAMP检测方法具有高特异度、高灵敏度、快速简便、可视化判读结果等优势,适合在基层进行H7N9人禽流感病毒的快速检测,并可以应用于大流感疫情的准确筛查。Objective A reverse transcriptase loop- mediated isothermal amplification assay was developed for establishing a specific,sensitive,rapid,visible detection method of H7N9 human avian influenza virus. Methods According to the conserved sequence of H7N9 human avian influenza virus HA gene,with the help of the software Primer Explorer V4,five primers specific to the seven site of HA gene were designed,reaction system and amplification conditions were optimized,and the specificity and sensitivity were verified. Results The H7N9 subtype influenza virus were detected and there was no amplification reaction with other subtypes of subtype H1,subtype H3,subtype H5,subtype H6,subtype H9 influenza virus,influenza virus B,parainfluenza,respiratory syncytial virus,coronavirus,metapneumovir,adenovirus and human bocavirus. The lowest detection limit was 0. 01 pg. In addition,the amplification could be finished within 45 min and with the addition of calcein,the presence of results could be detected by naked eyes. Conclusion The RT- LAMP assay developed in this study is simple and rapid and has the advantages of high specificity,high sensitivity,with visual interpretation of the results,which is suitable for rapid detection of H7N9 human avian influenza virus at the grassroots level,and can be applied to high flu accurate screening.

关 键 词：H7N9 人禽流感病毒 反转录-环介导等温扩增技术 基层检测 

分 类 号：S852.65[农业科学—基础兽医学]