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目的建立检测甲型肝炎减毒活疫苗病毒滴度的细胞培养/链特异性逆转录聚合酶链式反应(strand-specific reverse transcriptase-polymerase chain reaction,strand-specific RT-PCR)方法。方法根据甲型肝炎病毒(HAV)L-A-1株基因组序列,设计5条特异性引物。将甲型肝炎减毒活疫苗原液感染2BS细胞,收获增殖高峰期的HAV,提取RNA,进行2轮链特异性PCR扩增,检测HAV复制过程中的负链RNA,通过Reed-Muench公式计算HAV病毒滴度,并对建立的细胞培养/链特异性RT-PCR法进行特异性及敏感性验证,同时用该方法检测4批冻干甲型肝炎减毒活疫苗病毒滴度,并与《中国药典》三部(2010版)中甲型肝炎减毒活疫苗病毒滴度检测方法(细胞培养时间为28 d/ELISA法)进行比较。结果 HAV负链RNA经2轮扩增获得阳性扩增条带,大小与预期相符。该方法检测HAV复制过程中的负链RNA,特异性、敏感度均较好。两种方法检测的病毒滴度接近,差异无统计学意义(P>0.05)。结论建立的细胞培养/链特异性RT-PCR方法检测周期短,灵敏度、特异性好,可用于疫苗的滴度检测。
Objective To establish a strand-specific reverse transcriptase-polymerase chain reaction (strand-specific RT-PCR) method for detecting the titer of live attenuated hepatitis A virus vaccine. Methods According to the genomic sequence of Hepatitis A virus (HAV) L-A-1 strain, five specific primers were designed. 2BS cells were infected with live attenuated hepatitis A vaccine, HAVs were harvested at the peak of proliferation, RNA was extracted, 2-cycle specific PCR amplification was performed to detect negative-strand RNA during HAV replication, HAV was calculated by Reed-Muench formula Virus titers, and established cell culture / chain-specific RT-PCR method for specificity and sensitivity verification, using this method to detect four batches of freeze-dried hepatitis A attenuated live virus vaccine titer, and "China The attenuated live attenuated hepatitis A virus titre was tested in the Pharmacopoeia (2010 edition) (cell culture time 28 d / ELISA). Results The HAV negative-strand RNA amplified by two rounds positive amplification bands, the size of the expected. The method detects negative RNA in the process of HAV replication and has better specificity and sensitivity. The virus titer detected by the two methods was similar, with no significant difference (P> 0.05). Conclusion The established cell culture / strand specific RT-PCR method has short detection period, good sensitivity and specificity and can be used for the titration of vaccines.