细胞培养结合实时荧光RT-PCR法快速检测甲肝病毒滴度的研究

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8d检测病毒滴度为lg107.0CCID50/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(P0.05)。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。

Determination of hepatitis A vaccine by cell culture combined with TaqMan-based real-time RT-PCR

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 lgl07~CCIDs0/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 Determination of hepatitis A vaccine by cell culture combined with TaqMan-based real-time RT-PCR is sensitive, specific and rapid. It could be applied in detection of HAV in the vaccine production process.