杜贝病毒、盖勒尤卜病毒、蒂亚福拉病毒等三种内罗病毒实时荧光RT-PCR检测方法研究

Establishment of real-time RT-PCR methods for detection of Dugbe, Qalyub and Thiafora viruses in nairovirus genus

目的建立可检测杜贝病毒(dugbe virus,DUGV)、盖勒尤卜病毒(qalyub virus,QALV)和蒂亚福拉病毒(thiafora virus,TFAV)等三种内罗病毒基因组的实时荧光RT-PCR检测方法。方法收集、整理,比对、分析在公共数据库发布的三种病毒基因组S节段核苷酸序列,确定检测靶标,利用生物信息软件设计特异性引物、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检测方法均可有效扩增检测病毒靶标RNA,检测限分别为160 copies/μL,20 copies/μL和10 copies/μL,检测科萨努尔森林病病毒、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-刚果出血热病毒和塔西那病毒样本无非特异性扩增,三种内罗病毒相互间无交叉反应,重复性分析显示变异系数均在2%以内。结论本研究建立的检测DUGV、QALV和TFAV的实时荧光RT-PCR方法,可用于相关临床样本、媒介、宿主动物标本以及进出口物品筛查检测。

Objective To establish real-time fluorescent RT-PCR methods for detection of the three viruses in nairovirus genus,including dugbe virus(DUGV),qalyub virus(QALV)and thiafro virus(TFAV).Methods The S-segment sequences of the three virus genomes 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 using bioinformatic software.Real-time fluorescent quantitative RT-PCR detection methods were established with protocol optimization.The simulated samples prepared by in vitro transcription technique,specimens of infections with other viruses,virus strains and blood samples from healthy human were used to evaluate the detection limit,specificity and reproducibility of the methods.Results The established real-time fluorescent quantitative RT-PCR detection methods could effectively amplify and detect the viral target RNA,with detection limits of 160 copies/μL,20 copies/μL and 10 copies/μL,respectively.No nonspecific amplification in the samples of Kyasanur Forest disease virus,influenza BV and BY viruses and influenza A(H3N2)virus,yellow fever virus,encephalitis B virus,Crimean-Congo hemorrhagic fever virus,Tahyna virus.No cross reaction was detected among the three nairoviruses.The coefficient of variation was within 2%in the reproducibility analysis.Conclusions The real-time RT-PCR methods for the detection of DUGV,QALV and TFAV established in this study could be used for screening and detection of related clinical samples,vectors,host animal samples and import and export articles.