数字PCR技术检测rAAV产品中质粒DNA残留  

Detection of plasmid DNA residues in rAAV products by digital PCR technology

作　　者：凡文超 于雷[1] 安怡方 王光裕[1] 史新昌[1] 周勇[1] FAN Wen-chao;YU Lei;AN Yi-fang;WANG Guang-yu;SHI Xin-chang;ZHOU Yong(National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《药物分析杂志》2023年第12期2004-2009,共6页Chinese Journal of Pharmaceutical Analysis

基　　金：北京市科技计划资助(Z221100007922015)。

摘　　要：目的:利用数字PCR(dPCR)技术检测重组腺相关病毒(recombinant adeno-associated virus, rAAV)产品中质粒DNA残留。方法:针对质粒抗性基因KanR序列的4个不同区段设计引物探针,建立单重和双重dPCR检测KanR残留量;在双重dPCR中,双阳性孔代表2组引物对之间的序列完整,通过测试单、双阳性孔的比例,评价KanR序列的分布情况;利用DNaseⅠ消化游离的质粒DNA,评价病毒衣壳内错包装的质粒DNA情况。结果:4组单重dPCR实测KanR残留量之间基本一致(1.054×10^(8)~1.467×10^(8)copies·μL^(-1));双重dPCR与单重dPCR实测结果间无明显差异;双重dPCR在6~6 000 copies·μL^(-1)浓度范围内显示出良好的线性关系(r>0.999)和重复性(RSD<20%);1/2,2/3和3/43种组合的双重PCR的双阳性孔占比基本一致,表明KanR基因的断裂可能更趋向于随机断裂;DNaseⅠ处理前后结果提示,错包装的大片段质粒DNA(1/4双阳性)比游离的占比更高。结论:dPCR技术可用于检测rAAV产品中质粒DNA残留量,多重dPCR还可评估质粒DNA片段大小及分布情况。Objective:To detect plasmid DNA residues in recombinant adeno-associated virus(rAAV)products by digital PCR(dPCR).Methods:Primer probes were designed for four different segments of the plasmid resistance gene KanR sequence,and singleplex and duplex dPCR were established to detect KanR residues.In duplex dPCR,double positive wells represent the sequence integrity between two sets of primer pairs,and the distribution of KanR sequences was assessed by testing the proportion of single and double positive wells.The presence of incorrectly packaged plasmid DNA within the viral capsid was evaluated by digesting free plasmid DNA with DNaseⅠ.Results:The KanR residues measured in four groups by single dPCR were basically consistent(1.054×10^(8)-1.467×10^(8))copies·μL^(-1).There was no significant difference between the measured results of duplex dPCR and singleplex dPCR in each channel.It showed good linearity(r>0.999)and repeatability(RSD<20%)in 6-6000 copies·μL^(-1)concentration range for the established duplex dPCR.The proportion of double positive wells in the dual PCR of 1/2,2/3,and 3/43 combinations was basically consistent,indicating that the breakage of the KanR gene may tend to be more random.The results before and after DNaseⅠtreatment suggested that the proportion of large fragments of plasmid DNA(1/4 double positive)that are wrongly packaged was higher than that of free plasmid DNA.Conclusion:The dPCR technology can be used to detect plasmid DNA residues in rAAV products,and multiple dPCR can also evaluate the size and distribution of plasmid DNA fragments.

关 键 词：数字PCR 重组腺相关病毒 质粒DNA残留 KanR基因 

分 类 号：R917[医药卫生—药物分析学]