鸽圆环病毒Cap基因SYBR GreenⅠ荧光定量PCR方法的建立及应用

Development and Application of SYBR GreenⅠFluorescent Quantitative PCR for Detection of Cap Gene of Pigeon Circovirus

为建立一种针对鸽圆环病毒(PiCV)的快速诊断方法,试验根据PiCV的Cap保守基因序列,设计合成一对特异性引物,建立了PiCV荧光定量PCR检测方法,对其敏感性、特异性、重复性进行评价并利用该方法对临床样本进行检测。结果表明:在最佳反应条件下,所建立的PiCV荧光定量PCR方法在1×10^(2)~1×10^(7) copies/μL标准品范围内具有良好的线性相关性,线性相关系数为0.9846;敏感性试验结果显示,该方法最低可检测到1×10^(2) copies/μL标准品,是常规PCR检测方法的100倍;特异性试验结果显示,该方法与其他常见的鸽病病原不发生交叉反应;重复性评价结果显示,批内与批间的变异系数均小于2%;所建立PiCV荧光定量PCR方法对临床病死鸽肝脏样品检测结果显示,PiCV阳性率达75.7%,高于常规PCR方法的检测阳性率(54.1%),说明该方法具有良好的适用性。可应用于对PiCV的快速检测,为PiCV的及时防控提供有力技术支持。

In order to develop a rapid diagnostic method for pigeon circovirus(PiCV),a pair of specific primers were designed and synthesized according to the cap conservative gene sequence of PiCV for development of a fluorescence quantitative PCR method.Subsequently,this assay was evaluated for its sensitivity,specificity,repeatability and suitability for testing clinical samples.The results showed that under the optimal reaction conditions,this assay had a good linear correlation in the range of 1×10^(2)-1×10^(7 )copies/μL and the linear correlation coefficient of 0.9846.The minimum detection limit of standard substance was 1×10^(2) copies/μL,which was 100 times sensitive than that of the conventional PCR method.The method did not react with other common pigeon pathogens.Repeatability evaluation results showed that the coefficients of variation within and between batches were less than 2%.Then this method along with the conventional PCR was used to test liver samples of dead pigeons.The positive rates of PiCV were 75.7%for the fluorescence quantitative PCR method and 54.1%for conventional PCR.Taken together,this method developed in this study could be used as a better choice for rapid detection of PiCV and technical support for the timely prevention and control of PiCV.