Development,verification and application of fluorescence quantitative PCR method for detection of virus titer of recombinant measles virus vector SARS-CoV-2 vaccine
Objective To develop,verify and preliminarily apply a fluorescence quantitative PCR(qPCR)method for detec-tion 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 0.20 µmol/L,which specifically detected the copy number of SARS-CoV-2 S gene.The linear relationship was good(R2=0.995)at the template concentration ranged from 2 × 102to 2 × 108 copies/μL,and the limit of detection was 2 × 102 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.
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