荧光定量环介导逆转录等温扩增技术检测丙型肝炎病毒反应体系的建立

Development of hepatitis C virus by fluorescent real-time reverse transcription loop-mediated isothermal amplification method

目的建立一种快速、灵敏、特异性高的方法定量检测丙型肝炎病毒RNA。方法根据丙型肝炎病毒基因5’UTR序列中8个基因区段设计3对特异引物同时加入SYBRGreenⅠ染料在反转录酶和BstDNA聚合酶的作用下60℃等温条件下扩增1h后根据荧光曲线进行定量。结果120例经实时荧光定量PCR检测阳性的血清样品进行检测阳性符合率100％。110例健康体检者的血清经两种方法检测均阴性。对卫生部临检中心提供10’IU／ml的质控样本采用阴性血清稀释后进行灵敏度的实验，结果显示荧光定量环介导逆转录等温扩增（RT—LAMP）技术可检出10IU／ml浓度的RNA分子颗粒。结论成功建立了荧光定量环介导逆转录等温扩增技术检测丙型肝炎病毒的方法。恢技术具有快速、灵敏度高、特异性强的特点。

Objective To establish a rapid, sensitive, and specific quantitative method to detect hepatitis C virus. Methods A primer set targeting HCV 5＇UTR was designed. The isothermal amplification was performed by the Bst DNA polymerase and AMV reverse transcriptase, under the temperature of 60℃for 60 min. The signal was monitored by SYBR Green I . Results One hundred and twenty positive serum samples, confirmed by the real-time PCR. All were detected by the isothermal amplification, while 110 healthy subjects＇ samples were negative by the both methods. The lower detect limit was determined to 10 IU/ml HCV-RNA, by the assay on serial dilutions of the quality control standards obtained from clinical in- vestigation center of MOH. Conclusion A real time reverse loop-mediated isothermal amplification method was developed to detect HCV, with the characteristic of rapidity, high sensitivity and specificity.