实时荧光定量RT-PCR技术快速检测H5亚型禽流感病毒  

RAPID DETECTION OF AVIAN INFLUENZA VIRUS H5 BY USING REAL-TIME RT-PCR

作　　者：吕恒[1] 张锦海[1] 王长军[1] 顾海涛[1] 王平[1] 鲁娟东[1] 

机构地区：[1]南京军区疾病预防控制中心,南京210002

出　　处：《解放军预防医学杂志》2012年第6期398-401,共4页Journal of Preventive Medicine of Chinese People's Liberation Army

基　　金：江苏省科技支撑计划(社会发展)项目(No.BE2012609);国家重大传染病专项课题(No.2008ZX10004-008);国家"十一五"科技重大项目(No.2009ZX10004-315);南京军区重点课题(No.10Z038);南京军区重点课题(No.11C040)

摘　　要：目的建立快速检测H5亚型禽流感病毒HA基因的实时荧光定量RT-PCR方法,为禽流感病毒的监测和感染的预防控制提供科学的技术手段。方法针对H5亚型禽流感病毒血凝素(HA)基因保守区域设计特异性引物与荧光探针;通过体外克隆技术获得阳性定量标准品;优化探针、引物浓度、反应体系与反应条件,提高检测方法的特异性、敏感性与重复性,并进行定量分析。结果反应体系具有良好的扩增效率,80 min内可完成扩增检测工作;检测灵敏度为100拷贝/反应,通过外标准品建立的标准曲线在一条直线上;3个外标准品的变异系数(CV)分别为2.60%、2.47%、2.44%;H5亚型禽流感病毒培养液、确诊人感染高致病性禽流感患者标本为阳性扩增结果,而其它流感病毒临床标本及阴性对照均呈阴性。结论本研究建立的实时荧光定量RT-PCR方法具有敏感、快速、重复性好的特点,适合于H5亚型禽流感病毒的快速定量检测。Objective To establish a method for rapid detecting avain influenza virus subtype H5（AIV H5） by using real time reverse transcription-polymerase chain reaction（RT-PCR） assay,so as to provide scientific basis for prevention and control of highly pathogenic avian influenza.Methods A pair of specific primers and a fluorogen-labeled probe were designed and synthesized according to highly conserved regions of the hemagglutinin gene of AIV H5.Clone technology was used to gain a quantitative standard which been used for analysis.The reaction parameters such as the concentration of primers,probes and the reaction buffer were optimized to develop one set of real-time RT-PCR assay for rapid detection of AIV H5.Results The optimal experiment had good amplification efficiency,and could be completed within 80 min.A linear standard curve was obtained in the experiment,and the limit of the detection was 100 copies per reaction.The coefficient of variation（CV） of the three external standard were 2.6%,2.47%,2.44% respectively.The culture medium of AIV H5,and the specimen taken from human case of AIV H5 infection showed positive reactions;while other influenza viruses,and negative control no cross reaction.Conclusion The real-time RT-PCR detection method has high specificity,sensitivity,and reproducibility.It is suitable for rapid detection of avian influenza virus H5.

关 键 词：实时荧光定量RT-PCR H5亚型禽流感病毒(AIVH5) 血凝素基因 

分 类 号：R373.1[医药卫生—病原生物学]