磁珠法结合定量PCR检测肠道病毒71型灭活疫苗中宿主细胞DNA残留量的验证及应用

Validation and application for determination of residual host cell DNA in enterovirus 71 inactivated vaccine by magnetic bead based extraction combined with quantitative PCR

目的对磁珠法结合定量PCR（quantitative PCR，qPCR）检测肠道病毒71型灭活疫苗（Vero细胞）（EV71疫苗）中宿主DNA残留量进行适用性验证及应用。方法利用微磁珠与蛋白溶液中DNA的特异性结合，来提取样品中残留的宿主细胞DNA。将已知浓度的细胞DNA对照系列稀释后，作为标准品DNA与提取的DNA同时进行qPCR扩增。根据标准品DNA的循环阈值与浓度之间的线性关系，对未知样品中残留DNA进行定量分析。结果标准品检测范围在0．03～3000．00Pg／反应。该方法的标准曲线决定系数≥O．980，扩增效率为90．O％～110．00。质控样品回收率为50％～150％，相对标准偏差均小于30％。实验结果的各项参数均在要求范围内。结论磁珠法可解决残留DNA检测中样品前处理的技术难点，qPCR能够简便、快速、准确地对EVTl疫苗生产过程中的DNA残留量进行定量测定。该法适用于EV71疫苗中DNA残留量的检测及疫苗生产过程和其成品的质量控制，对其他采用同样细胞基质的病毒性疫苗质量控制具有借鉴意义。

Objective To verify the detection method for residual host cell DNA in enterovirus 71 inactivated vaccine （Veto cell） （EV7l vaccine） by magnetic bead based extraction combined with quantitative PCR （qPCR）. Methods Residual host cell DNA in samples was extracted utilizing the specific binding of magnetic bead to DNA in protein solution, qPCR was performed on extracted DNA and serial diluted standards simultaneously. Residual DNA in samples was quantitatively analyzed according to the linear relationship between cycle threshold values and concentrations of standards. Results The standard detection range was 0. 03-3 000. 00 pg/reaction. The correlation coefficient of standard curve was ：〉0. 980, with amplification efficiency at 90. 0%-110. 0%. The recovery rates of spiked samples were 50%-150%, and the relative standard deviation was 〈30%. All parameters of results were within the required range. Conclusions The magnetic bead based extraction method can solve technical difficulties in sample pretreatment for residual DNA assay, qPCR is a simple, rapid and accurate method for quantitation of residual DNA in EV7l vaccine. This method is suitable for quality control of EV7l vaccine in production,and may provide indication for quality control of other same cell based viral vaccines.