Sabin株脊髓灰质炎灭活疫苗Vero细胞DNA残留定量PCR检测法的验证

目的  验证Sabin株脊髓灰质炎灭活疫苗中Vero细胞DNA残留定量PCR（quantitative PCR，qPCR）检测法。方法  用试剂盒提取Sabin株脊髓灰质炎灭活疫苗原液的DNA，采用qPCR法检测Vero细胞DNA，并验证方法的线性、专属性、准确性、精密度、耐用性及定量限；同时采用DNA探针杂交法检测样品。结果  qPCR检测Vero细胞DNA标准曲线在0.003 0~300.000 pg/μl之间线性良好；对Hep-2细胞、大肠埃希菌、酵母细胞DNA均无扩增反应；高、中、低浓度样品加标回收率在50%~150%，准确性良好；定量检出限为0.003 0 pg/μl；精密度良好（相对标准偏差＜15%）；反应体系配制好后置于2~8 ℃ 30 min后进行检测，相对标准偏差＜15%，耐用性良好。DNA探针杂交法与qPCR结果均小于0.1 pg/μl。 结论  qPCR检测Sabin株脊髓灰质炎灭活疫苗中Vero细胞DNA残留具有良好的线性、专属性、准确性、精密度和耐用性，可适用于该项检测。

Validation of a quantitative PCR method forVero cell DNA residue inSabin poliomyelitis inactivated vaccine

Objective  To validate quantitative PCR (qPCR) method for Vero cell DNA residue detection in Sabin poliomyelitis inactivated vaccine (sIPV). Methods  DNA of sIPV was extracted by kit and Vero cell DNA residue was detected by qPCR. The linear, specificity, accuracy, precision, durability and quantitative limit of the method were validated. The samples were simultaneously detected by the DNA probe hybridization method. Results  The standard curve had good linearity (coefficient of determination at 0.999) in the range of 0.003 0-300.000 0 pg/μl DNA. The detection was not interfered by the genome DNA of Hep-2 cell, E. coli and yeasts. The recoveries of high, medium and low concentration samples were 50%-150%, showing good accuracy. The quantitation limit was 0.003 0 pg/μl. Precision was good, with relative standard deviation (RSD)＜15%. When the reaction system was placed at 2-8 ℃ for 30 minutes after prepared, durability results were good with RSD＜15%. The results of DNA probe hybridization method and qPCR were both less than 0.1 pg/μl. Conclusion  The qPCR method has good linearity, specificity, accuracy, precision, and durability, and can be applied to Vero cell DNA residue detection in sIPV.