Objective To establish a method based on PCR and restriction endonucleases for rapid identification of spike protein (S) genes of different strains in recombinant adenovirus SARS-CoV-2 vaccine,so as to use it for quality control of the vaccine.Methods Snap Gene 5.2 software was used to simulate and analyze the fragments of S gene sequences of SARS-CoV-2 wild-type (WT),Beta,Omicron and BA.5 variants digested by KpnⅠ,StuⅠ and BsmFⅠ.Specific primers were designed according to the conserved regions of S genes of the above strains,and PCR amplification was performed using the extracted recombinant adenovirus SARS-CoV-2 vaccine genomic DNA of each strain as the template to acquire S gene PCR products.The PCR products were digested separately with three restriction endonucleases,KpnⅠ,StuⅠand BsmFⅠ,and the size and number of the digested fragments were analyzed by 0.8% agarose gel electrophoresis.Different strains were mixed in pairs,and PCR amplification and restriction enzyme digestion were performed according to the above method to verify the ability of this method to identify different S genes in mixed strains.Results The results of software simulation analysis showed that after the PCR products of different strains were digested by KpnⅠ,StuⅠand BsmFⅠ,the sizes of restriction fragments were consistent with expectations,and there were obvious difference among different strains.The PCR product of WT strain vaccine could be digested into two fragments by KpnⅠ,but could not be digested by StuⅠor BsmFⅠ.The S gene of Beta strain vaccine could be digested into two fragments by StuⅠ,but could not be digested by KpnⅠand BsmFⅠ.The S gene of Omicron strain vaccine could be digested into two fragments by BsmFⅠ,but not by StuⅠand KpnⅠ.The S gene of BA.5 strain vaccine could be digested by KpnⅠand BsmFⅠat the same time,but not by StuⅠ.After different SARS-CoV-2 strains were mixed in pairs,it had no effect on the detection results of PCR and restriction enzyme combination method,and the S genes of different strains could be accurately identified.Conclusion The established method based on PCR and restriction enzymes can quickly and accurately identify the S genes of different strains in recombinant adenovirus SARS-CoV-2 vaccine,which provides an effective means for the quality control of the vaccine.