基于细胞感染的实时定量逆转录PCR检测水痘减毒活疫苗病毒滴度方法的建立  

Establishment of a cell infection-based real time quantitative reverse-transcription PCR for virus titer of live attenuatedvaricellavaccine

作　　者：王玉霞 徐然 毛辉 谢蕾 王亮 陈晓 周旭 朱为 Wang Yuria;Xu Ran;Mao Hui;Xie Lei;Wang Liang;Chen Xiao;Zhou Xu;Zhu Wei(No.3 Research Laboratory,Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 200051,China;Vaccine DivisionⅢ,Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 201403,China;Department of Quality Control,Shanghai Institute of Biological Products Co.,Ltd.Shanghai 201403,China;Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 201403,China)

机构地区：[1]上海生物制品研究所有限责任公司第三研究室,上海200051 [2]上海生物制品研究所有限责任公司疫苗三室,上海201403 [3]上海生物制品研究所有限责任公司质量检定室,上海201403 [4]上海生物制品研究所有限责任公司,上海201403

出　　处：《国际生物制品学杂志》2023年第1期50-54,共5页International Journal of Biologicals

摘　　要：目的建立基于细胞感染的实时定量逆转录PCR(real time quantitative reverse-transcription PCR, RT-qPCR)法, 用于水痘减毒活疫苗感染性病毒滴度检测。方法根据水痘减毒活疫苗毒种V-Oka株的基因序列设计特异性引物及探针。将参考品及待测样品系列稀释后接种MRC-5细胞, 经培养后收获细胞, 提取RNA, 进行一步法RT-qPCR检测, 通过平行线法计算样品滴度, 并将检测结果与中国药典2020年版三部规定的空斑法进行对比。结果感染细胞适宜收获时间为36~48 h, 可检测样品滴度范围为2.8~5.0 lgPFU/ml。与空斑法检测的滴度之间的差异无统计学意义(冻干疫苗组t=1.09, 疫苗原液组t=0.59, P值均>0.05)。结论初步建立了基于细胞感染的RT-qPCR法, 可用于水痘减毒活疫苗病毒滴度检测。Objective To establish a cell infection-based real time quantitative reverse-transcription PCR(RT-qPCR)method for infectious virus titration of live attenuated varicella vaccine.Methods Specific primers and probe were designed according to the gene sequence of live attenuated varicella vaccine strain V-Oka.Confluent MRC-5 cell monolayers were inoculated with serial dilutions of reference and test samples.After incubation for certain time,cells were harvested,from which RNA was extracted and applied to one-step RT-qPCR reaction.Titers of test samples were calculated by parallel line analysis,and compared with results by plaque method according to Chinese pharmacopeia 2020 edition volume Il.Results The suitable harvest time of varicella-zoster virus infected cells was 36-48 h,and the detectable titers ranged from 2.8-5.0 lgPFU/ml.Titers detected by RT-qPCR and plaque method had no statistically significant difference(t=1.09 for lyophilized vaccine and t=0.59 for vaccine bulk,both P>0.05).Conclusion The cell infection-based RT-qPCR method is preliminarily established,which can be applied to titrate infectious virus in live attenuated varicella vaccine.

关 键 词：实时定量逆转录聚合酶链反应 水痘减毒活疫苗 滴度 

分 类 号：R511.[医药卫生—内科学]