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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 establish an efficient and simple real-time fluorescence quantitative PCR method for the determination of recombinant adenovirus titer in AdEasy system. Methods: Stratagene AdEasyTM vector system was used to construct recombinant adenovirus in 293 cells. Ten times diluted virus stock solution was used to extract genomic DNA and plaque assay. Real-time PCR reactions were performed with a 10-fold gradient of pAdEasy plasmid as a standard template to amplify hexon gene fragments and draw a standard curve. Then the above recombinant adenovirus DNA as a template, using the same system for real-time PCR reaction, while using agarose plaque assay viral stock titer. Results: The recombinant adenovirus was successfully constructed and plaque assay was performed. The PCR reaction using standard templates showed that the linear range of this method was 101 to 108 copies. The DNA copy concentration (vg / ml) of the virus stock solution is approximately 10 times the value of the plaque titer (pfu / ml). Conclusion: The real-time quantitative PCR method can detect the recombinant adenovirus titer in a large linear range, which reflects the actual quantity of recombinant adenovirus more accurately than the plaque assay.