| 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 |
| |
| 引用本文： | 张静静 宋路萍 安文琪 马小伟 梁雪爽 郭冰峰 荆新蕊 马超援 张少宁 毕利利 王斌. 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[J]. 国际生物制品学杂志, 2018, 41(3): 123-127. DOI: 10.3760/cma.j.issn.1673-4211.2018.03.005 |
| |
| 作者姓名： | 张静静 宋路萍 安文琪 马小伟 梁雪爽 郭冰峰 荆新蕊 马超援 张少宁 毕利利 王斌 |
| |
| 作者单位： | 453000 新乡，华兰生物工程股份有限公司研发中心 |
| |
| 基金项目： | “重大新药创制”科技重大专项(2014ZX09102042-003) |
| |
| 摘 要： | 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〔非洲绿猴肾细胞（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Vero细胞DNA。将已知浓度Vero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0.03～3 000.00 pg/反应。该方法的标准曲线决定系数≥0.98，扩增效率在90%～110%。质控样品回收率在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、准确地对生产过程样品的EV71疫苗中DNA残留量进行定量测定。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，对其他以Vero细胞为基质的病毒性疫苗质量控制具有借鉴意义。
|
| 关 键 词： | 定量聚合酶链反应 Vero细胞 DNA残留 磁珠法 宿主细胞 |
Validation and application of residual host cell DNA determination in enterovirus 71 inactivated vaccine (Vero cells) by magnetic bead based extraction combined with quantitative PCR |
| |
| Affiliation: | Research and development center, Hulan Biological Eengineering Inc., Xinxiang 453000, China |
| |
| Abstract: | Objective To verify the detection method for residual host cell DNA in enterovirus 71 inactivated vaccine [African green monkey cell (Vero cell)] (EV71 vaccine) by magnetic bead based extraction combined with quantitative PCR (qPCR). Methods Residual Vero 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 Vero DNA standards simultaneously. The amount of residual DNA in samples was quantitatively analyzed according to the linear relationship between threshold circle values and concentrations of DNA standards. Results The standard DNA detection range was 0.03-3 000.00 pg/reaction. The correlation coefficient of standard curve was >0.98, with PCR amplification efficiency at 90%-110%. The recovery rate of spiked samples was 50%-150%, and the relative standard deviation was <30%. All parameters of the experimental 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 Vero cell DNA in EV71 vaccine. Thus, it is suitable for quantity control of EV71 vaccine in production. This result may provide indication for quality control of other Vero cell based viral vaccines. |
| |
| Keywords: | Quantitative polymerase chain reaction Vero cells Residual DNA Magnetic bead based extraction Host cell |
|
| 点击此处可从《国际生物制品学杂志》浏览原始摘要信息 |
|
点击此处可从《国际生物制品学杂志》下载免费的PDF全文 |
