Validation and preliminary application of VSV-G qPCR assay for detection replication competent lentivirus
10.3760/cma.j.cn112309-20201215-00553
- VernacularTitle:复制型慢病毒VSV-G qPCR检测方法的验证及初步应用
- Author:
Xueling WU
1
;
Xiang ZHAO
;
Shufang MENG
Author Information
1. 中国食品药品检定研究院生物制品检定所细胞资源保藏研究中心，卫生部生物技术产品检定方法及其标准化重点实验室，北京 100050
- Keywords:
Replication competent lentivirus (RCL);
VSV-G;
Chimeric antigen receptor T cell (CAR-T cell)
- From:
Chinese Journal of Microbiology and Immunology
2021;41(7):538-544
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish VSV-G qPCR assay for detection of replication competent lentivirus(RCL) and verify its application.Methods:A real-time fluorescent quantitative PCR for VSV-G envelope gene was developed. Several parameters including specificity, linear, amplification efficiency, precision, trueness, dynamic range, limit of detection, limit of quantification and robustness were verified. Preliminary application on CAR-T cells, end of production cells and the harvest of lentivirus vector was performed by using the method developed.Results:The real-time fluorescent quantitative PCR assay for VSV-G was specific for the detection VSV-G without specific amplification on 293T, PBMC and C8166 cells. The linear range of the assay was 1×10 2 copies/test-1×10 9 copies/test with a R2 value more than 0.998 and amplification efficiency between 93% and 98%. The precision (relative standard deviation) of the assay was less than 12% and the trueness (the rate of recovery) of the assay was between 85% and 106%. The limit of detection (LOD) and limit of quantification (LOQ) of the assay was 5 copies/test and 40 copies/test. In addition, the robustness of the assay was also well. All the results of validation illustrated that the assay could meet the detection requirements. All of the 54 samples including CAR-T cells, lentivirus vector and end of product cells after amplification and passage on C8166 cells were negative of RCL by using the established assay. Conclusions:The real-time fluorescent quantitative PCR for VSV-G were established successfully. All of the validation results illustrated that the assay could meet the detection requirements. The application of the assay was conducive to further enhance the safety of the lentivirus vector related products.
