扎伊尔型埃博拉病毒的实时荧光RT-PCR检测方法研究

Development of Real-time RT-PCR assay for detection of Zaire Ebolavirus

目的 建立扎伊尔型埃博拉病毒的核酸检测方法,以期用于埃博拉出血热临床标本的检测.方法 针对扎伊尔型埃博拉病毒核蛋白和糖蛋白基因设计引物和探针,建立单重和双重实时荧光RT-PCR检测方法,利用体外转录病毒RNA和埃博拉病毒系列参考品RNA评价其敏感性,利用马尔堡病毒、健康人、登革热患者和发热伴血小板减少综合征患者血清评价其特异性.结果 所建立的实时荧光RT-PCR检测方法扩增效率在95％～105％,可特异性地检测扎伊尔型埃博拉病毒核蛋白和糖蛋白基因,与马尔堡病毒、登革热和发热伴血小板减少综合征病毒均无交叉反应,体外转录的病毒RNA可检出10～100拷贝/μl.双重检测方法通过细胞培养的扎伊尔型埃博拉病毒RNA验证,可检出100 pfu/ml病毒.结论 本研究建立的检测扎伊尔型埃博拉病毒的实时荧光RT-PCR方法具有良好的特异性和敏感性,可用于埃博拉出血热临床标本的检测.

Objective To establish a method for Zaire Ebolavirus （ZEBOV） RNA detection and laboratory diagnosis of suspected ZEBOV infected cases.Methods Primers/probe sets for Nucleoprotein （NP） and Glycoprotein（GP） detection were designed and used to develop monoplex and duplex real-time RT-PCR assays.The sensitivity was evaluated by in vitro transcribed RNA and ZEBOV RNA reference,and the specificity was identified by Marburg virus,healthy human sera and sera from Dengue fever patients and Severe fever with thrombocytopenia syndrome （SFTS） patients.Results The developed real-time RT-PCR assay scould be used for ZEBOV NP and GP detection specifically,and the amplification efficiency was 95% -105%.There was no cross reaction with Marburg virus,Dengue fever virus and SFTS virus.The sensitivity was evaluated with serial dilutions of synthesized viral RNAs,and the results showed that in vitro transcribed RNA of 10-100 copies/μl could be detected.The developed duplex assay was further verified with extracted RNA of ZEBOV collected from cell culture,and viruses in titer of 100 pfu/ml could be successfully detected.Conclusion Real-time RT-PCR assays for ZEBOV detection were established in this study,and proved to be specific and sensitive,which can be used for laboratory detection of suspected human Ebola hemorrhagic fever cases.