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。

abstractsObjective: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 SARS-CoV-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 0.032 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.