犬圆环病毒EvaGreen实时荧光定量PCR检测方法的建立与初步应用

Establishment and preliminary application of EvaGreen real-time fluorescence quantitative PCR for detection of canine circovirus

建立了犬圆环病毒(canine circovirus,CanineCV)EvaGreen实时荧光定量PCR检测方法。用PCR方法克隆CanineCVRep基因中的高保守区,扩增的目的片段大小为217bp,将其克隆至pMD18-T载体上,构建PCR检测阳性参考质粒,并用阳性参考质粒,进行各项反应条件优化。建立的标准曲线呈现出较高拟合性的线性回归,R2值为0.9995;该方法检测灵敏度为3.88×10^(1) copies/μL;在重复性试验中,组内和组间重复变异系数均小于2%,表明该方法具有良好的重复性;该方法对犬瘟热病毒、犬细小病毒、犬流感病毒、犬副流感病毒、犬腺病毒2型、犬冠状病毒等犬常见病原的检测均无交叉反应;临床样本检测表明,所建立的EvaGreen实时荧光定量PCR对CanineCV的阳性检出率为2.82%(5/177),高于普通PCR检测的CanineCV阳性率1.13%(2/177)。本研究成功建立了一种用于快速检测CanineCV的EvaGreen qPCR方法,该方法安全且具有较好的特异性、敏感性和重复性,可作为CanineCV感染的病原学检测工具。

The aim of this study is to establish an EvaGreen real-time fluorescent quantitative PCR method for detection of canine circovirus.The highly conserved region in Repgene of canine circovirus was cloned by PCR,and the amplified target fragment was 217bp in size,then it was cloned into pMD18-T vector.A positive reference plasmid for PCR detection was thus constructed,and was used to optimize the reaction conditions.The established standard curve showed a high fitting linear regression with a correlation coefficient of 0.9995.The detection sensitivity of this method was 3.88×10^(1) copies/μL.In the repeatability test,the coefficient of variation within and between groups were all less than 2%,which indicates that this method had good repeatability.This method had no cross reaction to canine distemper virus,canine parvovirus,canine influenza virus,canine parainfluenza virus,canine adenovirus type 2and canine coronavirus.The detection of clinical samples showed that the positive detection rate of canine circovirus by EvaGreen real-time fluorescence quantitative PCR was 2.82%(5/177),which was higher than that of canine circovirus detected by ordinary PCR 1.13%(2/177).In this study,we successfully established an EvaGreen qPCR method for rapid detection of canine circovirus,which is safe,specific,sensitive and repeatable,and can be used as a detection tool for canine circovirus infection.