TaqMan实时荧光定量RT-PCR检测甲型肝炎减毒活疫苗病毒含量的研究

摘要

目的：建立一种快速定量检测甲型肝炎减毒活疫苗病毒含量的实时荧光定量RT-PCR方法。方法对Gen-Bank中登陆的甲型肝炎减毒活疫苗株（ L-A-1）和其他甲型肝炎病毒基因组全序列比较分析，根据其高度保守的5′端非编码区设计针对甲型肝炎减毒活疫苗株特异性引物与探针，对荧光定量RT-PCR反应条件进行优化，检测该方法的特异性和灵敏性，并对甲型肝炎减毒活疫苗病毒含量进行定量检测。结果该方法对甲型肝炎减毒活疫苗株高度特异，扩增片段为207 bp，不与其他肠道病毒发生非特异性反应。在104 CCID50/管～10-1 CCID50/管之间有良好的扩增曲线，检测的灵敏度可达0．1CCID50～0．01CCID50，比普通RT-PCR高100倍。结论该方法具有快速、灵敏、特异、重复性好等优点，可应用于甲型肝炎减毒活疫苗生产过程中病毒含量滴度测定及指导疫苗成品的配制。%Objective To establish a specific TaqMan-based Real-time RT-PCR assay for the rapid detection of hepatitis A virus content in hepatitis A ( live) vaccine ( HAV) .Methods The gene in attenuated strain of hepatitis A ( L-A-1) con-tained in HAV was down-loaded from Genbank and aligned by using the biologic software , and the specific primers and probes were designed in the 5′-NCR of L-A-1.The primers and probes as well as the reaction condition were optimized to improve the sensitivity and specificity for the assay .And then this method was used in detection of RNA for hepatitis A virus in HAV.Results A 207 bp fragment was specifically amplified from L-A-1 strain, but not from the tested other enterovir-uses.It had no cross reaction with all the other enteroviruses .The sensitivity of this assay was 0.1 CCID50 .The amplifica-tion curve was in a linear correlation in between 104 CCID50 /tube-10-1 CCID50/tube.Conclusion The TaqMan-based real-time RT-PCR assay established in this study was sensitive and precise for the specific and rapid detection of hepatitis A virus content in HAV .It was applied successfully in preparation of the vaccine product in process of HAV production .