Q-PCR法检测腺病毒基因治疗产品中的复制型腺病毒

摘要

目的:建立Q-PCR法检测腺病毒基因治疗产品中的复制型腺病毒(RCA).方法:首先制备高纯度的pXC1质粒参考品(含有E1基因序列),紫外吸收法定量后经过数学换算得到拷贝数;然后比较染料法和探针法的灵敏度,建立适用的Q-PCR检测方法;最后用新建Q-PCR法定量测定腺病毒基因治疗产品中的RCA.结果:pXC1质粒参考品的拷贝数初步标定为2.6×1010拷贝/μL;探针法的灵敏度比染料法高1000倍;将pXC1质粒参考品应用于Q-PCR(探针法)测定腺病毒基因治疗产品中的RCA,标准曲线回归方程为y=-1.416ln (x) +47.395,R2=0.9966,供试品的Cq值均位于标准曲线范围内.结论:新建Q-PCR法适用于检测腺病毒基因治疗产品中的RCA,且结果准确可靠.%Objective:To develop a Q-PCR method to detect replication-competent adenoviruses(RCA) in adenoviral gene therapy products.Methods:Highly purified pXC1 plasmid(containing E1 gene) reference was prepared,the potency was determined by UV method,and the copy number was calculated by mathematical transformation.The sensitivity of SYBR Green method and probe method were compared to determine the appropriate Q-PCR.RCA in adenoviral gene therapy products were detected by newly established Q-PCR method.Results:The copy number of pXC1 plasmid reference was preliminarily calibrated to be 2.6×1010 copies per μL;the sensitivity of probe method was 1000 times higher than SYBR Green method.pXC1 plasmid reference was applied to detect RCA in adenoviral gene therapy products by Q-PCR,the regression equation was y=-1.416ln(x)+47.395,R2=0.9966,and the Cq values of test samples were located within the scope of standard curve.Conclusion:The developed Q-PCR with high sensitivity is suitable to detect RCA in adenoviral gene therapy products,and its accuracy and reliability are validated.