CAJ | 학술논문

目的：建立一种细胞培养与实时荧光RT-PCR相结合的快速检测甲肝病毒滴度的方法。方法根据甲肝病毒（ HAV） L-A-1株5′端基因组序列，设计了2条基因特异性引物及一条探针，建立实时荧光RT-PCR法，结合细胞培养检测甲肝病毒滴度，并与ELISA检测法进行比较。结果实验中建立的方法能特异检测甲肝病毒，细胞培养8 d检测病毒滴度为lg107．0 CCID50/mL。同一样本重复检测3次，批内样本Ct值的变异系数最大为0．89％，批间样本Ct值变异系数最大为1．66％。建立的细胞培养结合实时荧光RT-PCR法（细胞培养8 d）与细胞培养ELISA法（细胞培养28 d）检测甲肝病毒滴度结果差异无统计学意义（P＞0．05）。结论该方法具有快速、灵敏、特异等优点，应用于疫苗常规检测有良好前景。
목적：건립일충세포배양여실시형광RT-PCR상결합적쾌속검측갑간병독적도적방법。방법근거갑간병독（ HAV） L-A-1주5′단기인조서렬，설계료2조기인특이성인물급일조탐침，건립실시형광RT-PCR법，결합세포배양검측갑간병독적도，병여ELISA검측법진행비교。결과실험중건립적방법능특이검측갑간병독，세포배양8 d검측병독적도위lg107．0 CCID50/mL。동일양본중복검측3차，비내양본Ct치적변이계수최대위0．89％，비간양본Ct치변이계수최대위1．66％。건립적세포배양결합실시형광RT-PCR법（세포배양8 d）여세포배양ELISA법（세포배양28 d）검측갑간병독적도결과차이무통계학의의（P＞0．05）。결론해방법구유쾌속、령민、특이등우점，응용우역묘상규검측유량호전경。
Objective To establish a method for the rapid detection of hepatitis A virus by cell culture combined with Taq-Man-based Real-time RT-PCR.Methods The specific primers and probe were designed in the 5′-NCR of HAV (L-A-1). Cell culture combined with TaqMan-based real-time RT-PCR was used in determination of HAV , and compared with detec-tion by ELISA.Results The developed method is able to detect hepatitis A virus after HAV infected 2BS cells.The titer of HAV reaches lg107.0CCID50/mLas the HAV infected 2BS cells at 8th day, The coefficients of variation (CV) of intra-and inter-assay are calculated and they were less than 0.89% and 1.66% respectively,when the detection of a sample is re-peated for three times .Cell culture combined with TaqMan-based real-time RT-PCR is used in determination of hepatitis A vaccine, and compared with detection by ELISA , which has no statistical significance (P>0.05).Conclusion Determi-nation of hepatitis A vaccine by cell culture combined with TaqMan-based real-time RT-PCR is sensitive ,specific and rap-id.It could be applied in detection of HAV in the vaccine production process .