阿瓦朗病毒和休斯病毒实时荧光RT-PCR检测方法研究  

Real-time fluorescent quantitative RT-PCR methods for detection of Avalon virus and Hughes virus

作　　者：杜珊珊 李阿茜 黄晓霞[1] 刘洋[1] 王芹[1] 李川[1] 梁米芳[1] 李德新[1] 王世文[1] 李建东[1] Du Shanshan;Li Aqian;Huang Xiaoxia;Liu Yang;Wang Qin;Li Chuan;Liang Mifang;Li Dexin;Wang Shiwen;Li Jiandong(Key Laboratory of Biosafety,National Health and Family Planning Commission,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会生物安全重点实验室,北京102206

出　　处：《中华实验和临床病毒学杂志》2021年第1期111-115,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：国家科技重大专项(2018ZX10711001,2018ZX10101-002,2016ZX10004222-002)。

摘　　要：目的建立可以检测阿瓦朗病毒(Avalon virus,AVAV)和休斯病毒(Hughes virus,HUGV)两种内罗病毒的实时荧光定量RT-PCR检测方法,并进行初步的评价。方法收集、整理、比对、分析在公共数据库发布的两种病毒基因组核苷酸序列,确定检测靶标,设计特异性引物、探针,优化检测程序,建立实时荧光定量RT-PCR检测方法。利用体外转录技术制备的模拟样本、其他病毒感染标本、病毒株和正常人血标本比较评价所建方法的检测限、特异性、重复性特征。结果所建实时荧光定量RT-PCR检测方法可有效扩增检测AVAV和HUGA靶标RNA,检测限分别约为20拷贝/μl和70拷贝/μl,检测科萨努尔森林病毒、乙型流感病毒BV和BY型、甲型流感病毒H3N2、黄热病毒、乙型脑炎病毒、克里米亚-刚果出血热病毒、发热伴血小板减少综合征、内罗毕羊病毒和塔西那病毒样本无非特异性扩增,两种内罗病毒相互间无交叉反应,重复性比较分析显示变异系数小于2%。结论本研究建立的检测AVAV和HUGV的实时荧光定量RT-PCR方法,可用于临床样本检测和媒介生物、宿主动物标本筛查,便于病原的快速识别和疾病诊断。Objective To establish real-time fluorescent quantitative RT-PCR method for detection of Avalon virus(AVAV)and Hughes virus(HUGV),two Nairoviruses in the family of Nairoviridae.Methods The genomic sequences of the two viruses published in the international public database were collected,collated,compared and analyzed to define the detection targets,and the viral specific primers and probes were designed accordingly.Real-time fluorescent quantitative RT-PCR detection method were established,and the operating detection procedures were optimized using simulated samples prepared using in vitro transcription assay,other virus infected samples,virus strains and normal human blood samples.The detection limit,specificity and repeatability of the method were evaluated.Results The real-time fluorescent quantitative RT-PCR method could effectively amplify and detect AVAV and HUGV viral target RNA with detection limits of about 20 copies/μl and 70 copies/μl,respectively.No nonspecific amplification was found in the samples of Kyasanur forest disease virus,influenza B virus BV and BY,influenza A virus H3N2,yellow fever virus,Japanese encephalitis virus,Crimean-Congo hemorrhagic fever virus,SFTS virus,nairobi sheep disease virus and Tahyna virus.There was no cross reaction between the two nairoviruses.The coefficient of variation was within 2%by repeated comparative analysis.Conclusions The real-time fluorescent quantitative RT-PCR method for detection of AVAV and HUGV might be used for screening of humans,vectors and host animal samples for rapid detection of related pathogens.

关 键 词：内罗病毒 实时荧光RT-PCR 阿瓦朗病毒 休斯病毒 

分 类 号：R440[医药卫生—诊断学]