Establishment and verification of a digital PCR detection method for genome titer of adeno-associated virus gene therapy products
10.13200/j.cnki.cjb.004275
- VernacularTitle:腺相关病毒基因治疗产品基因组滴度数字PCR检测方法的建立及验证
- Author:
AN Yifang
- Publication Type:Journal Article
- Keywords:
Digital polymerase chain reaction(dPCR);
Adeno-associated virus(AAV);
Genome titer;
Gene therapy
- From:
Chinese Journal of Biologicals
2024;37(8):897-903+910
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo establish and preliminarily verify a digital polymerase chain reaction(dPCR)method for the detection of genome titer of adeno-associated virus(AAV)gene therapy products.MethodsThrough dPCR detection of the same batch of AAV gene therapy products,the primers,probes,sample treatment methods and concentrations of primer and probe were optimized. The specificity,linearity,relative accuracy and repeatability of the optimized method were verified,and the limit of quantitation(LOQ)and detection range were determined.ResultsNFS-05 vector titer 5.0 crop primer probe was selected,the final primer concentration was 800 nmol/L,the final probe concentration was 400 nmol/L,and the sample pretreatment method was digestion by Proteinase K. The established method had good specificity. The correlation coefficient(R2)of the standard curve was greater than 0. 999 in the range of 3. 3 × 10~5-1. 3 × 10~8vg/mL. The relative accuracy was between 70% and 130%,the CV of repeatability was not more than 15%,and the LOQ was 3. 3 × 10~5vg/mL.ConclusionThe established dPCR method has good specificity,linearity,accuracy and repeatability,and can be applied to the determination of the genome titer of NFS-05 recombinant adeno-associated virus(rAAV)stock solution products.
关键词(KeyWords)：数字PCR;腺相关病毒;基因组滴度;基因治疗
Digital polymerase chain reaction(dPCR);Adeno-associated virus(AAV);Genome titer;Gene therapy
基金项目(Foundation): 北京市科技计划课题资助（Z221100007922015）
作者(Author): 安怡方,毕华,刘家池,魏云欢,赵琴,于雷,史新昌,裴德宁,李响,周勇,秦玺,梁成罡
AN Yifang,BI Hua,LIU Jiachi ,WEI Yunhuan ,ZHAO Qin ,YU Lei,SHI Xinchang,PEI Dening,LI Xiang,ZHOU Yong,QIN Xi,LIANG Chenggang
