CAJ | 학술논문

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。
목적：건립일충쾌속정량검측갑형간염감독활역묘병독함량적실시형광정량RT-PCR방법。방법대Gen-Bank중등륙적갑형간염감독활역묘주（ L-A-1）화기타갑형간염병독기인조전서렬비교분석，근거기고도보수적5′단비편마구설계침대갑형간염감독활역묘주특이성인물여탐침，대형광정량RT-PCR반응조건진행우화，검측해방법적특이성화령민성，병대갑형간염감독활역묘병독함량진행정량검측。결과해방법대갑형간염감독활역묘주고도특이，확증편단위207 bp，불여기타장도병독발생비특이성반응。재104 CCID50/관～10-1 CCID50/관지간유량호적확증곡선，검측적령민도가체0．1CCID50～0．01CCID50，비보통RT-PCR고100배。결론해방법구유쾌속、령민、특이、중복성호등우점，가응용우갑형간염감독활역묘생산과정중병독함량적도측정급지도역묘성품적배제。
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 .