Establishment and validation of droplet digital PCR assay for detection of template DNA residues in SARS-CoV-2 mRNA vaccine
10.13200/j.cnki.cjb.004461
- VernacularTitle:新型冠状病毒mRNA疫苗模板DNA残留数字微滴PCR检测方法的建立及验证
- Author:
ZHAO Danhua
- Publication Type:Journal Article
- Keywords:
SARS-CoV-2;
mRNA vaccine;
Template DNA residues;
Droplet digital PCR(ddPCR);
Quality control
- From:
Chinese Journal of Biologicals
2025;38(04):442-449
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish and validate a droplet digital PCR(ddPCR) method for the detection of template DNA residues in SARS-CoV-2 mRNA vaccine, thereby providing technical support for the quality control of SARS-CoV-2 mRNA vaccine.Methods With the replicon of the common DNA sequence of different plasmids as the target gene, specific primers and probe were designed to establish a ddPCR method for detecting the template DNA residues in SARS-CoV-2 mRNA vaccine. The established method was validated for linear range, repeatability, accuracy, intermediate precision, robustness,limit of detection(LOD), limit of quantification(LOQ), and specificity. The validated ddPCR method was subsequently applied to detect template DNA residues in SARS-CoV-2 mRNA vaccines targeting various variants from different manufacturers.Results The optimal annealing temperature for the established ddPCR was determined to be 58 ℃. The method demonstrated good linearity within a copy number range of 19 to 4 729 copies/μL, with a correlation coefficient(R2) of 0. 999 9.The repeatability coefficient of variation(CV) was less than 10%, and the accuracy recovery rate ranged from 99% to 121%,with a CV of less than 10%. Both CVs of intermediate precision and robustness exhibited less than 10%. The LOD and LOQ of the method were 9 copies/μL and 26 copies/μL, respectively, with good specificity. When applied to detect template DNA residues in SARS-CoV-2 mRNA vaccines from four different manufacturers targeting various variants, the CVs were all not more than 15%, and the recovery rates ranged from 79% to 138%.Conclusion The established ddPCR method for detecting template DNA residues in SARS-CoV-2 mRNA vaccine demonstrates high sensitivity, excellent stability, strong specificity, and high accuracy, making it a suitable approach for the detection of template DNA residues in SARS-CoV-2mRNA vaccine.
