探针实时荧光定量PCR检测白藜芦醇体外抗鸭瘟病毒活性方法的建立及应用

建立以TaqMan荧光探针为特点的PCR,检测白藜芦醇体外抗鸭瘟病毒活性。本研究针对鸭瘟病毒UL30基因序列设计特异性引物和TaqMan荧光探针,建立鸭瘟病毒TaqMan探针实时荧光定量PCR检测方法,并验证方法的特异度、敏感度和稳定性及对临床样品进行检测;运用该方法检测白藜芦醇对鸭胚成纤维细胞接种鸭瘟病毒后细胞中病毒增殖情况的影响,用以评价该药物体外抗鸭瘟病毒效果。结果显示,该方法建立的定量标准曲线阈值循环数（Threshold cycle,Ct）与模板拷贝数呈良好线性关系（R2=0.997）,斜率为-3.328,扩增效率（E）为99.7%,具有良好的特异度、敏感度和稳定性并能对临床样本进行准确的检测。运用该方法检测白藜芦醇体外抗鸭瘟病毒结果显示：经白藜芦醇处理后,染毒细胞中病毒拷贝数为（7.71±0.37）×10^4,与病毒对照组比较（5.63±0.26）×10^6明显减少（P〈0.01）。结果表明,TaqMan探针实时荧光定量PCR方法建立并成功用于体外药物抗鸭瘟病毒的检测;白藜芦醇具有良好的体外抗鸭瘟病毒活性。

Development and application of real-time fluorescence quantitative PCR for detecting the antiviral activity of resveratrol against duck plague virus in vitro

We developed a TaqMan probe real-time fluorescence quantitative PCR assay for detecting the antiviral activity of resveratrol against duck plague virus（DPV） in vitro. At first,primers and probe specific to UL30 gene of DPV were designed. A TaqMan probe real-time fluorescence quantitative PCR was established and its specificity, sensitivity and stability were assessed. The amounts of DPV in duck embryo fibroblasts（DEFs） treated with or without resveratrol were detected by this method. A fine linear relationship between the threshold cycle（Ct） values of the developed standard curve and the copy number of template was observed（R2 =0. 997）. The amplifica- tion efficiency of assay was 99.7% ,and the slope was-3. 328. This approach has good specificity, sensitivity and stability. In the application, the amounts of the DPV in the DEFs were detected successfully. The results showed that the amounts of the DPV in the DEFs treated with resveratrol was （7.71±0.37）× 10^4 ,which is significant difference with the amounts of the DPV in the DEFs treated without resveratrol（5. 63±0. 26） × 10^6. The results indicated the fluorescence quan- titation PCR based on TaqMan probe for detecting the DNA of DPV is successfully developed and the resveratrol showed the good ability to inhibit the DPV multiplication.