鼠源性病毒实时荧光定量PCR检测方法的建立及验证  

Development and validation of real-time fluorescence quantitative PCR method for murine virus

作　　者：王吉[1] 王莎莎[1] 李威[1] 付瑞[1] 谭淑萍 范婷婷 郑立群 刘志坚 汤华东 万滔 岳秉飞[1] WANG Ji;WANG Shasha;LI Wei;FU Rui;TAN Shuping;FAN Tingting;ZHENG Liqun;LIUZhijian;TANG Huadong;WAN Tao;YUE Bingfei(National Institutes for Food and Drug Control,National Center for Quality of Laboratory Animal,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院国家实验动物微生物遗传检测中心,北京102629 [2]舒泰神(北京)生物制药股份有限公司,北京100176 [3]未名生物医药有限公司,福建厦门361009 [4]武汉海特生物制药股份有限公司,湖北武汉430056

出　　处：《中国生物制品学杂志》2023年第11期1361-1367,1372,共8页Chinese Journal of Biologicals

基　　金：国家科技支撑计划(2015BAI09B02);国家药典委员会“药品医疗器械审评审批制度改革专项课题”(ZG2018-3-02)。

摘　　要：目的建立8种鼠源性病毒的实时荧光定量PCR(real-time fluorescent quantitative PCR,Q-PCR)检测方法,并进行验证。方法通过4个实验室验证Q-PCR法的特异性、灵敏度及精密性,同时进行病毒模拟污染试验及盲样检测,并将检测结果进行比对。采用Q-PCR法检测26批SARS-CoV-2疫苗生产用单抗细胞株及15批其他鼠源性制品。结果用于8种鼠源病毒检测的Q-PCR法与同科同属或其他鼠源病毒无交叉反应;除实验室2对鼠痘病毒(又名脱脚病病毒)(ectromelia virus,EctV/Mouse Pox,MPV)检测的灵敏度为2×10^(2)copies/μL外,实验室2对其他7种病毒及其他3个实验室对8种鼠源性病毒的检测灵敏度均为2×10^(1)copies/μL;除实验室3对鼠腺病毒(mouse adenovirus,MAdV)检测拷贝数试验间CV为37.58%外,实验室3对其他7种病毒和其他3个实验室对8种病毒检测的试验内、试验间Ct和拷贝数CV均<25%。病毒模拟污染试验检测灵敏度均符合参数要求;4个实验室盲样检测结果符合率为100%。26批SARS-CoV-2疫苗生产用单抗细胞株及15批其他鼠源性制品8种鼠源病毒检测结果均为阴性。结论鼠源性病毒的Q-PCR法具有良好的特异性、灵敏度及精密性,可用于鼠源性生物制品的检测。Objective To develop and validate the real-time fluorescent quantitative PCR(Q-PCR)method for the detection of 8 murine viruses.Methods The specificity,sensitivity and precision of the Q-PCR method were verified by four laboratories,and the virus simulated contamination test and blind sample detection were carried out simultaneously,of which the detection results were compared.The Q-PCR method was used to detect 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other products of murine origin.Results The Q-PCR method used for detecting 8 kinds of murine viruses had no cross reaction with the same family and genus or other murine viruses.Except the sensitivity of laboratory 2 to ectromelia virus(EctV/Mouse Pox,MPV)was 2×10^(2)copies/μL,the sensitivity of laboratory 2 to other 7 viruses and 3 other laboratories to 8 murine viruses was 2×10^(1)copies/μL.Except the inter-assay CV of the copy number of mouse adenovirus(MAdV)detected by laboratory 3 was 37.58%,the intra-assay and inter-assay CVs of the Ct and copy number of other 7 viruses detected by laboratory 3 and those of 8 viruses detected by other 3 laboratories were all less than 25%.The sensitivity of virus simulated contamination test met the parameter requirements.The coincidence rate of blind sample detection results by 4 laboratories was 100%.All the 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other murine derived products were negative for 8 murine viruses.Conclusion Q-PCR method for murine virus has good specificity,sensitivity and precision,and can be used for the detection of murine derived biological products.

关 键 词：鼠源性病毒 荧光定量PCR法 方法验证 

分 类 号：R-33[医药卫生]