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作 者:周艳萍 卢佳 李茜 林凤杰 杨安纳 孟胜利 王泽鋆 申硕 ZHOU Yan-ping;LU Jia;LI Qian;LIN Feng-jie;YANG An-na;MENG Sheng-li;WANG Ze-jun;SHEN Shuo(Wuhan Institute of Biological Products Co.,Ltd,Wuhan 430207,Hubei Province,China)
机构地区:[1]武汉生物制品研究所有限责任公司,湖北武汉430207 [2]国家联合疫苗工程技术研究中心,湖北武汉430207
出 处:《中国生物制品学杂志》2022年第6期711-717,共7页Chinese Journal of Biologicals
基 金:国家重点研发计划(2020YFC0842100)。
摘 要:目的对磁珠法结合实时定量PCR(real-time quantitative PCR,qPCR)检测新型冠状病毒(简称新冠)灭活疫苗(Vero细胞)中宿主细胞残留DNA(residual cell DNA,rcDNA)进行验证及应用。方法利用磁珠与溶液中DNA的特异性结合来提取样品中rcDNA。将已知浓度的细胞DNA标准品系列稀释后,根据DNA标准品的循环阈值与浓度之间的线性关系,对未知样品中的rcDNA进行定量分析。对方法进行线性和范围、专属性、准确度、精密度验证,并使用该方法对新冠灭活疫苗(Vero细胞)生产过程样品进行rcDNA及其片段大小检测。结果标准品检测范围为0.0003~30 pg/μL,该方法的标准曲线决定系数(R;)>0.99,扩增效率为90%~110%。质控样品回收率为50%~150%,相对标准偏差(RSD)均小于30%。试验结果的各项参数均符合标准。结论磁珠法可解决rcDNA检测中样品前处理提取DNA的技术难点,qPCR能够简便、快速、准确地对新冠疫苗生产过程中的rcDNA进行定量测定。该方法适用于新冠疫苗中rcDNA的检测及疫苗生产过程和成品的质量控制,对其他采用同样细胞基质的病毒性疫苗质量控制也具有借鉴意义。Objective To validate and apply an assay of residual host cell DNA in an inactivated SARS-CoV-2 vaccine(Vero cells)by magnetic beads-based extraction combined with real-time quantitative PCR(qPCR).Methods Residual cell DNA(rcDNA)in samples was extracted based on the specific binding of DNA in solution and magnetic beads.The standard cell DNA at known concentration was 10-fold serially diluted,and the rcDNA in samples at unknown concentration was quantitatively analyzed based on the linear relationship between cycle threshold value and concentration of standard.The developed method was validated for linear range,specificity,accuracy and precision,by which the rcDNA and the lengths of its fragments in samples in production process of inactivated SARS-CoV-2 vaccine(Vero cells)were determined.Results The quantitative range of standard was 0.0003~30 pg/μL.The correlation coefficient(R2)of standard curve was more than 0.99,while the amplification efficiency was 90%~110%.The recovery rates of spiked samples were 50%~150%,with a relative standard deviation of less than 30%.All the parameters of assays met the relevant requirements.Conclusion The magnetic beads-based DNA extraction overcame the technical difficulties in sample preparation for rcDNA assay.The qPCR is a simple,rapid and accurate method for quantitative determination of rcDNA in the production process of inactivated SARS-CoV-2 vaccine.The developed method is suitable for the determination of rcDNA and quality control of production process and final product of inactivated SARS-CoV-2,which provides a reference for the quality control of other viral vaccines prepared with the same cell matrix.
关 键 词:新型冠状病毒灭活疫苗 Vero细胞 宿主细胞残留DNA 实时定量聚合酶链反应 磁珠法
分 类 号:R37[医药卫生—病原生物学]
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