新型冠状病毒Beta株灭活疫苗原液鉴别实验  

Identification of SARS-CoV-2 Beta strain inactivated vaccine bulk

作　　者：庞德钦 周艳萍 杨安纳 周以斯 杨洁 杨东升 吴杰 王泽鋆 郭靖 申硕 Pang Deqin;Zhou Yanping;Yang Anna;Zhou Yisi;Yang Jie;Yang Dongsheng;Wu Jie;Wang Zejun;Guo Jing;Shen Shuo(Laboratory 1 of Viral Vaccine Research,Wuhan Institute of Biological Products Co.,Ltd.,National Engineering Technology Research Center for Combined Vaccines,Wuhan 430207,China)

机构地区：[1]武汉生物制品研究所有限责任公司病毒性疫苗研究一室,国家联合疫苗工程技术研究中心,武汉430207

出　　处：《国际生物制品学杂志》2023年第1期7-12,共6页International Journal of Biologicals

基　　金：国家重点研发计划(2020YFC0842100);湖北省科技重大专项(2021ACB005)。

摘　　要：目的建立特异性鉴别新型冠状病毒Beta株灭活疫苗原液的逆转录PCR(reverse transcription PCR, RT-PCR)并验证。方法在新型冠状病毒Beta株刺突蛋白(spike, S)受体结合域(receptor binding domain, RBD)中3个特异性突变位点的上下游保守区域设计引物S3F/S3R, 通过RT-PCR扩增843 bp目的DNA。对扩增产物进行测序, 与原型株的S基因序列进行对比, 通过3个特征突变识别Beta株。对该方法的重复性、专属性、耐用性和灵敏度进行验证。结果该方法能准确扩增出843 bp目的DNA, 经测序表明, 扩增产物与新型冠状病毒Beta株基因序列一致, 包含3个特征突变位点。取1批Beta株原液进行6次PCR, 测序结果显示与Beta株核苷酸序列一致。S3F/S3R引物只对S的RBD区域有扩增作用。Beta株原液4 ℃和-70 ℃放置8周, 仍然可以扩增出目的条带, 且测序结果正确。将原液提取RNA稀释成不同浓度后进行RT-PCR, 得出最低检测限为0.032 ng/μl。结论建立的RT-PCR具有良好的专属性、重复性、耐用性和灵敏度, 能成功区分新型冠状病毒原型株与Beta株, 可用于新型冠状病毒疫苗生产中原液的鉴别。Objective To establish and validate a reverse transcription PCR(RT-PCR)method for the specific identification of inactivated severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)Beta strain vaccine bulk.Methods Primers S3F/S3R were designed based on the sequence of upstream and downstream conserved regions to 3 specific mutation sites in the receptor binding domain(RBD)of spike(S)gene of Beta strain,and an 843 bp target DNA band was amplified by RT-PCR.The amplified bands were sequenced and compared with the S gene sequence of prototype strain.The Beta strain was identified by three characteristic mutations.The repeatability,specificity,durability and sensitivity of the method were verified.Results Target DNA bands of 843 bp were amplified,identical to the Beta strain gene sequence and containing 3 characteristic mutations.A batch of Beta strain bulk was taken for PCR 6 times,and the sequencing results showed that the nucleotide sequence was consistently identical to that of Beta strain.S3F/S3R primers only amplified the RBD region of SARSCoV-2 S.The target bands could still be amplified when the original solution of Beta strain was kept at 4℃or-70℃,respectively,for 8 weeks,and the sequencing results were consistent.The extracted RNA was diluted into different concentrations for RT-PCR,and the minimum detection limitation was O.032 ng/μl.Conclusions The established method has good specificity,repeatability,durability and sensitivity and can successfully distinguish SARS-CoV-2 prototype strain from Beta strain.It can be used for the identification of vaccine bulk in the production of COVID-19 vaccine.

关 键 词：新型冠状病毒Beta株 逆转录聚合酶链反应 鉴别 

分 类 号：Q503[生物学—生物化学]