基于叠氮溴化丙锭-定量反转录-聚合酶链式反应的甲型肝炎疫苗病毒滴度快速检测方法  

Rapid detection of virus titers of hepatitis A vaccine based on PMA-qRT-PCR

作　　者：卞莲莲[1] 王一平[1] 孙世洋[1] 高帆[1] 吴星[1] 毛群颖[1] 梁争论[1] BIAN Lian-lian;WANG Yi-ping;SUN Shi-yang;GAO Fan;WU Xing;MAO Qun-ying;LIANG Zheng-lun(National Institutes for Food and Drug Control,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院,北京102629

出　　处：《中国病毒病杂志》2020年第6期421-425,共5页Chinese Journal of Viral Diseases

基　　金：国家“十三五”重点研发计划(2017YFC1600703);国家“十三五”重大新药创制科技重大专项(2018ZX09101-001);国家药典委员会药品标准制修订研究课题(2019S006)。

摘　　要：目的建立快速检测甲型肝炎减毒活疫苗感染性病毒滴度的叠氮溴化丙锭-定量反转录-聚合酶链式反应(PMA-qRT-PCR)方法。方法比较叠氮溴化丙锭(PMA)浓度、光解时间、孵育温度和时间,以及不同灭活方式、pH值和添加剂预处理对PMA-qRT-PCR法检测的影响;进行线性、平行性、灵敏度、重复性、相关性、特异性和初步适用性研究。结果确定最适反应条件为PMA终浓度50μmol/L,4℃避光孵育30 min,光解30 min,病毒液pH值为1.48~7.43。方法学研究结果显示,该方法特异性良好;线性范围为5.0~8.0 lgCCID50/ml,线性和平行性良好;定量下限为5.0 lgCCID50/ml;重复性变异系数(CV)为2.78%~3.58%;与法定方法检测结果具有显著相关性(相关系数R2=0.698)。初步适用性结果显示,4个实验室使用各自细胞系检测同一样品结果差异无统计学意义(P=0.071~0.997);不同企业生产的疫苗在不同细胞系上检测适用性良好。结论建立了甲型肝炎减毒活疫苗感染性病毒滴度的PMA-qRT-PCR方法,为冻干甲型肝炎减毒活疫苗的效力评价提供了快速检测方法。Objective To establish a propidium monoazide-quantitative reverse transcription-polymerase chain reaction(PMA-qRT-PCR) method for the rapid detection of infective virus titers of live attenuated hepatitis A vaccine. Methods The effects of PMA concentration, photolysis time, incubation temperature, incubation time, different inactivation methods, pH value and additive pretreatment on the detection of hepatitis A virus by PMA-qRT-PCR were compared;then the linearity, parallelism, sensitivity, repeatability, correlation, specificity and preliminary applicability were studied. Results The optimal reaction conditions were as follows: the final concentration of PMA was 50 μmol/L, incubated at 4 ℃ in dark for 30 min, photolysis for 30 min;the pH values of virus solution were 1.48-7.43.The results showed high specificity of PMA-qRT-PCR;The linear range was 5.0-8.0 lgCCID50/ml, and exhibited good parallelism and linear correlation;the lowest detection limit of quantification was 5.0 lgCCID50/ml;the CV values of the repeatability were 2.78%-3.58%;it correlated significantly with the detection results of legal methods(correlation coefficient=0.698).The preliminary applicability results showed that there were no significant differences among the four laboratories using their own cell lines to detect the same samples(P=0.071-0.997);the vaccines produced by different manufacturers demonstrated good applicability in different cell lines. Conclusions A PMA-qRT-PCR method for the detection of infective virus titers of live attenuated hepatitis A vaccine is established.It provides a rapid detection method for the evaluation of the efficacy of live attenuated hepatitis A vaccine.

关 键 词：甲型肝炎疫苗 叠氮溴化丙锭 PMA PMA-qRT-PCR 病毒滴度 快速检测 疫苗效力 疫苗质量控制 

分 类 号：R373.2[医药卫生—病原生物学]