应用实时荧光定量PCR测定AdEasy系统中重组腺病毒滴度(英文)

Determination of recombinant adenovirus titer in AdEasy system with real-time taqman PCR assay

目的 :确立一种高效、简便的荧光实时定量PCR方法 ,对AdEasy系统中的重组腺病毒滴度进行测定。方法 :采用Stratagene公司的AdEasyTM载体系统在 2 93细胞中构建重组腺病毒 ,经 10倍梯度稀释的病毒母液用以提取基因组DNA及空斑测定。以 10倍梯度稀释的pAdEasy质粒作为标准模板进行实时PCR反应扩增六邻体基因片段 ,并绘制标准曲线。然后以上述的重组腺病毒DNA为模板 ,采用同样体系进行实时PCR反应 ,同时用琼脂糖空斑法测定病毒母液的滴度。结果 :成功构建了重组腺病毒并对其进行了空斑测定。运用标准模板进行的PCR反应显示该方法的线性范围为 10 1～ 10 8拷贝。病毒母液的DNA拷贝浓度(vg/ml)值 ,约为空斑滴度 (pfu/ml)值的 10倍。结论 :荧光实时定量PCR方法可在较大的线性范围内检测重组腺病毒滴度 ,较之空斑法更为准确地反映了重组腺病毒的实际数量。

Objective:To develop a real-time PCR assays based on TaqMan chemistry for the determination of physical titers of recombinant adenovirus of AdEasy system and to compare with plaque assay.Methods:The recombinant adenovirus was produced using AdEasy TM adenoviral vector system. A 10-fold series diluted primary viral stocks were used for plaque assay and DNA extraction. Plasmid pAdEasy-1 were 10-fold series diluted and served as standards. Real-time PCR amplification of the hexon gene was performed in triplicate for each diluted recombinant virus. At the same time,plaque assays were performed using overlay agarose method.Results:The standard linear(10 1 to 10 8 copies) from quantitation was achieved with each dilutions of plasmid. The precise quantities of each diluted recombinant virus were calculated by comparation with the standard curve. We also find that the 'vg/ml' titer value is generally about 10 times than 'pfu/ml' titer of the same recombinant virus stock.Conclusion:A TaqMan real-time PCR method is established to determine the 'vg/ml' titer of the recombinant adenovirus. The method is rapid and quantitative over a wide range of vector titers.