Omicron株新型冠状病毒灭活疫苗(Vero细胞)原液特异性鉴别试验RT-PCR法的建立及验证

Development and validation of RT-PCR method for specific identification test on bulk of inactivated vaccine against Omicron strain of SARS-CoV-2(Vero cells)

目的建立Omicron株新型冠状病毒灭活疫苗(Vero细胞)原液特异性鉴别试验的RT-PCR法,并对该方法进行验证。方法根据GIAIDS数据库中新型冠状病毒Omicron株及其他突变株和原型株的S基因序列差异设计并合成引物OSTF1、OS1R,提取Omicron株新型冠状病毒灭活疫苗(Vero细胞)原液病毒RNA,逆转录为cDNA,利用对Omicron株S蛋白基因序列中的特异性插入及缺失核苷酸序列设计的引物对进行PCR扩增,扩增产物进行1%琼脂糖凝胶电泳鉴定。对建立的RT-PCR法进行重复性、中间精密度、专属性、耐用性及灵敏度验证,并用该方法鉴别6批Omicron株新型冠状病毒灭活疫苗原液。结果Omicron株新型冠状病毒灭活疫苗(Vero细胞)原液利用引物OSTF1/OS1R进行RT-PCR检测,可扩增出约460 bp的条带,而原型株、Beta株、Delta株新型冠状病毒灭活疫苗原液(Vero细胞)均无法扩增出条带;重复3次检测、2名实验人员分别在3 d重复3次检测、利用3种不同退火温度进行RT-PCR,Omicron株新型冠状病毒灭活疫苗(Vero细胞)原液均可扩增出约460 bp的条带;原液蛋白浓度在0.04μg/mL以上、RNA浓度在0.128 ng/μL以上均可扩增出较亮的约460 bp的条带;6批Omicron株新型冠状病毒灭活疫苗(Vero细胞)原液经RT-PCR检测,均可见约460 bp的条带。结论RT-PCR法专属性、重复性、中间精密度、耐用性和灵敏度均良好,可用于Omicron株新型冠状病毒灭活疫苗(Vero细胞)原液鉴别。

Objective To develop and validate a RT-PCR method for the specific identification test on bulk of inactivated vaccine against Omicron strain of SARS-CoV-2(Vero cells).Methods Specific primers OSTF1 and OS1R were designed and synthesized by comparison of the sequence of SARS-CoV-2 Omicron variant with those of other mutant and prototype strains in GIAIDS database,by which the viral RNA was extracted from the bulk of inactivated vaccine against Omicron strain(Vero cells)and reversely transcripted into cDNA.A specific fragment of S protein gene of Omicron variant was amplified by PCR using the primers designed according to the insertion and deletion regions of Omicron S gene,and identified by 1%agarose gel electrophoresis.The developed RT-PCR method was validated for reproducibility,intermediate precision,specificity,durability and sensitivity,by which six batches of bulk of inactivated vaccine against Omicron strain(Vero cells)were identified.Results A 460 bp band was amplified from the bulk of inactivated vaccine against Omicron strain by RT-PCR using primers OSTF1/OS1R,while no bands were amplified from those against prototype,Beta and Delta strains.The 460 bp bands were amplified from the bulk of inactivated vaccine against Omicron strain(Vero cells)in three repeated tests,the tests repeated for 3 times within 3 d by two testers and RT-PCR at three temperatures for annealing.However,bright 460 bp bands were amplified at a protein concentration of more than 0.04μg/mL and a RNA concentration of more than 0.128 ng/μL in the bulk.The bands were amplified from all the six batches of bulk of inactivated vaccine against Omicron strain(Vero cells).Conclusion RT-PCR shows good specificity,reproducibility,intermediate precision,durability and sensitivity,which may be used for the identification of bulk of inactivated vaccine against Omicron strain(Vero cells).