Sabin株脊髓灰质炎灭活疫苗Vero细胞残余DNA荧光定量PCR检测方法及其相应质量标准的建立  

Establishment of real-time quantitative PCR method for determination of residual Vero cell DNA in inactivated poliovirus vaccine made from Sabin strain and corresponding quality standard

作　　者：江征[1] 刘悦越[1] 朱文慧 沈泓 李炎 郭航炜 王剑锋[1] 英志芳[1] 王晓娟[4] 李长贵[1] JIANG Zheng;LIU Yue-yue;ZHU Wen-hui;SHEN Hong;LI Yan;GUO Hang-wei;WANG Jian-feng;YING Zhi-fang;WANG Xiao-juan;LI Chang-gui(National Institutes for Food and Drug Control,Beijing 102629,China;不详)

机构地区：[1]中国食品药品检定研究院,北京102629 [2]浙江省食品药品检验研究院,浙江杭州310052 [3]四川省药品检验研究院,四川成都611731 [4]国家药典委员会,北京100061

出　　处：《中国生物制品学杂志》2022年第8期981-985,991,共6页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2018ZX09737-003);国家药典委员会药品标准提高课题(2020S001).

摘　　要：目的建立与国际接轨的Sabin株脊髓灰质炎灭活疫苗(inactivated poliovirus vaccine made from Sabin strain,sIPV)Vero细胞残余DNA荧光定量PCR(q-PCR)检测方法及相应质量标准。方法按照《中国药典》三部(2020版)通则3407第三法,选择通则建议的Vero细胞核酸引物和探针,通过优化磁珠法提取核酸以及PCR反应体系和条件,采用q-PCR法Vero细胞DNA定量国家标准品,对sIPV产品进行线性范围、精密度和回收率验证。在此基础上,与国内5家sIPV生产研究企业和2家省药检院协作开展适用性验证的研究,并对检测结果进行统计分析,建立q-PCR法检测sIPV成品Vero细胞DNA残留量的可接受限度标准。结果优化的q-PCR方法在0.002~200 pg/μL范围内呈良好的线性关系,R2=0.999;精密度验证结果试验内、试验间变异系数分别为16.7%和27%;样品回收率在50%~150%范围内,RSD≤30%,符合《中国药典》三部(2020版)通则3407第三法相关要求。协作研究各企业sIPV成品Vero细胞DNA残留量平均为0.07~7.8 pg/剂,检测结果最大值<50 pg/剂,各实验室显示出良好的适用性;用q-PCR法检测已知50 pg Vero细胞核酸标准品(杂交法用),共获得55个有效检测值,统计计算结果为50.00 pg(95%CI:44.00~56.01 pg),与理论值差异无统计学意义(P>0.05)。结论成功建立了sIPV产品适用的Vero细胞残余DNA q-PCR检测方法,该方法具有良好的适用性,可用于sIPV成品的质量评价,限度标准定为不高于50 pg/剂具有可行性。Objective To establish a real-time quantitative PCR(q-PCR)method for determination of residual Vero cell DNA in inactivated poliovirus vaccine made from Sabin strain(sIPV)as well as the corresponding quality standard.Methods According to the method 3 in General Rule 3407 of Chinese Pharmacopoeia(VolumeⅢ,2020 edition),the primers and probe for Vero cell nucleic acid were selected,the nucleic acids were extracted by optimized magnetic bead extraction method,and the reaction system and condition for PCR were optimized.The developed q-PCR method was verified for linear range,precision and recovery rate in determination of sIPV by using the national standard for quantitative determination of residual Vero cell DNA.On the basis of this,the suitability of the method was cooperatively verified by five manufacturers of sIPV and two provincial institutes for drug control,and the results were analyzed statistically to establish the standard for acceptable range of residual Vero cell DNA content in final product of sIPV determined by q-PCR.Results The linear range of optimized q-PCR method was 0.002~200 pg/μL with a R2 value of 0.999.The coefficients of variation(CVs)of results in intra-and inter-assays of verification for precision were 16.7%and 27%respectively.The recovery rate of samples was 50%~150%,with a RSD of not more than 30%,which met the relevant requirements in General Rule 3407 of Chinese Pharmacopoeia(VolumeⅢ,2020 edition).The mean residual Vero cell DNA content in final products of sIPV from various manufacturers was 0.07~7.8 pg/dose,while the maximum was less than 50 pg/dose,which showed good applicability.The Vero cell nucleic acid standard at a known content of 50 pg by hybridization method was determined by q-PCR,and the statistical result of 55 valid values was 50.00(95%CI:44.00~56.01)pg,which showed no significant difference with the theoretical value(P>0.05).Conclusion The q-PCR method for determination of residual Vero cell DNA in sIPV was successfully developed,which showed good suitability and m

关 键 词：Sabin株脊髓灰质炎灭活疫苗 VERO细胞 残余DNA 荧光定量PCR 

分 类 号：R392-33[医药卫生—免疫学]