摘要
目的建立检测重组麻疹病毒载体严重急性呼吸综合征冠状病毒2(severe acute respiratory symptom coronavirus 2,SARS-CoV-2)疫苗病毒滴度的荧光定量PCR(qPCR)法,并进行验证及初步应用。方法以重组质粒pUC57-S351为模板,扩增SARS-CoV-2 S基因,优化引物浓度,建立qPCR检测方法。对该方法的专属性、重复性进行验证,确定线性范围及检测限。应用建立的qPCR法对生物反应器连续培养感染后1~12 d的重组病毒S基因拷贝数进行检测。结果选择引物浓度为0.20μmol/L建立qPCR方法。该方法能特异性检测SARS-CoV-2 S基因拷贝数;模板浓度在2×10^(2)~2×10^(8)copies/μL之间,线性关系良好(R^(2)=0.995),检测下限为2×10^(2)copies/μL;6次重复检测重组病毒S基因拷贝数的变异系数(coefficient of variation,CV)为2.64%。应用建立的qPCR法检测生物反应器连续培养感染后1~12 d的重组病毒S基因拷贝数的结果与细胞病变法检测结果基本一致。结论建立的qPCR法专属性和重复性良好,可用于重组麻疹病毒载体SARS-CoV-2疫苗病毒滴度检测。
Objective To develop,verify and preliminarily apply a fluorescence quantitative PCR(qPCR)method for detection of the virus titer of recombinant measles virus vector severe acute respiratory symptom coronavirus 2(SARS-CoV-2)vaccine.Methods SARS-CoV-2 S gene was amplified using recombinant plasmid pUC57-S351 as the template,and the primer concentration was optimized to develop the qPCR detection method.The specificity and repeatability of the method were verified,and the linear range and limit of detection were determined.The copy number of recombinant virus S gene was detected by the developed qPCR method 1~12 d after continuous culture in bioreactor.Results The qPCR method was developed with the primer concentration of 0.20μmol/L,which specifically detected the copy number of SARS-CoV-2 S gene.The linear relationship was good(R^(2)=0.995)at the template concentration ranged from 2×10^(2)to 2×10^(8) copies/μL,and the limit of detection was 2×10^(2)copies/μL;The coefficient of variation(CV)value of 6 repeated detections of copy number of recombinant virus S gene was 2.64%.The copy number of recombinant virus S gene was monitored by the developed qPCR method 1~12 d after continuous culture in bioreactor,and the results were basically consistent with those detected by cytopathic method.Conclusion The developed qPCR method has good specificity and repeatability,which can be used for virus titer detection of recombinant measles virus vector SARS-CoV-2 vaccine.
作者
杜天飞
容新宗
杨盛理
张勇侠
高雅丽
武志强
李春阳
刘兰军
DU Tianfei;RONG Xinzong;YANG Shengli;ZHANG Yongxia;GAO Yali;WU Zhiqiang;LI Chunyang;LIU Lanjun(Chengdu Institute of Biological Products Co.,Ltd.,Center for Sichuan Province Vaccine Engineering Research,Chengdu 610023,Sichuan Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2024年第6期745-750,共6页
Chinese Journal of Biologicals
基金
成都市科技局重大科技创新项目(2020-YF08-00001-GX)。
作者简介
通信作者:刘兰军,E-mail:liulanjun@sinopharm.com;共享第一作者:杜天飞;共享第一作者:容新宗。
