小鼠微小病毒Q-PCR检测方法在病毒去除工艺验证中的应用

Application of Q-PCR method for murine minute virus in validation of procedure for virus clearance

目的将小鼠微小病毒(murine minute virus,MVM)Q-PCR检测方法应用于病毒去除工艺验证中。方法使用MVM Q-PCR法和细胞病变法同时检测10倍系列稀释后的MVM,建立核酸拷贝数与病毒滴度之间的函数关系,并应用于纳米膜过滤和层析病毒去除工艺验证中。结果核酸拷贝数与病毒滴度间呈线性关系,R^2可达0.9863。应用于纳米膜过滤去除工艺验证,两种方法检测结果具有一致性;应用于层析去除工艺验证,两种方法在亲和层析、阴离子柱层析、阴离子Q膜层析工艺中结果具有一致性,但在具有灭活病毒的疏水层析中,结果不一致。结论在病毒去除工艺验证中,MVM Q-PCR法可作为现有细胞病变法的补充。

Objective To apply the Q-PCR method for murine minute virus(MVM)to the validation of procedure for virus clearance.Methods The 10-fold serially diluted MVM was determined by MVM Q-PCR and CPE method simultaneously,based on which a functional relationship between nucleic acid copy number and virus titer was established and applied to the validation of procedure for virus clearance by nanomembrane filtration and chromatography.Results The copy number of nucleic acid was linearly correlated to the virus titer,with a R^2 value of 0.9863.The determination results by MVM Q-PCR and CPE method were in agreement in validation of virus clearance by nanomembrane filtration.However,in validation of various chromatographic procedures,the determination results by the two methods were in agreement in affinity chromatography,anion exchange chromatography and anion Q membrane chromatography,while were inconsistent in hydrophobic chromatography.Conclusion MVM Q-PCR method may be used as a supplement of CPE method for validation of procedure for virus clearance.