牛纽布病毒TaqMan荧光定量RT-PCR检测方法的建立及应用

Establishment and application of TaqMan real-time quantitative RT-PCR for detection of bovine nebovirus

牛纽布病毒(bovine nebovirus,BNeV)是国内近几年发现的引起犊牛腹泻的新病原,为了建立快速、准确且能定量分析BNeV的检测方法,本试验根据GenBank上发布的BNeV聚合酶基因(RdRp)序列设计合成了1对特异性引物和1条探针,并通过优化反应体系,成功建立了BNeV TaqMan荧光定量RT-PCR检测方法并对临床样品进行检测。结果表示,该方法最佳上下游引物浓度均为500 nmol/L,探针浓度为400 nmol/L,在1×10^(8)~1×10^(1)拷贝/μL之间呈现良好的线性关系,线性相关系数R^(2)=0.996,扩增效率为10^(5).9%;该方法特异性较强,在多个犊牛腹泻相关病原中,只检测出BNeV;敏感性较高,对BNeV质粒标准品最低检测下限为1×10^(1)拷贝/μL,而普通PCR对BNeV质粒标准品最低检测下限为1×10^(3)拷贝/μL;重复性较好,组内变异系数和组间变异系数均小于3%;对2021年3-5月采自内蒙古地区牧场的37份犊牛粪样中BNeV的检出率为35.1%,通过标准曲线计算其病毒载量,其中腹泻粪样平均病毒载量为8.7×10^(5)拷贝/μL。本研究建立的BNeV TaqMan荧光定量RT-PCR检测方法的特异性强、稳定性好、灵敏度高,为BNeV的检测和分子流行病学调查提供了有力的手段。

Bovine nebovirus(BNeV)is a new pathogen that causes diarrhea in calves discovered in China in recent years.In order to establish a rapid,accurate and quantitative analysis method for BNeV,the TaqMan fluorescent quantitative RT-PCR detection method for BNeV was successfully established using a pair of specific primers and a probe designed based on the BNeV polymerase gene(RdRp)published on GenBank and used to detect the clinical samples.The results showed that the optimal concentrations for upstream and downstream primer were 500 nmol/L,and 400 nmol/L for the probe with a good linear relationship between 1×10^(8)-1×10^(1)copies/μL,linear correlation coefficient R^(2)=0.996,and the amplification efficiency of 105.9%.This method had strong specificity,detecting only BNeV among multiple diarrhea-related pathogens in calves.The sensitivity was high with a minimum detection limit for BNeV plasmid standard of 1×10^(1)copies/μL in comparison with the lowest detection limit of BNeV plasmid standard by common PCR of 1×10^(3)copies/μL.The repeatability was good,with less than 3%of variation coefficient both in the intra-and inter-group.The detection rate of BNeV in 37 calf fecal samples collected from pastures in Inner Mongolia from March to May 2021 was 35.1%.The viral load calculated by the standard curve for the diarrhea fecal samples was 8.7×105 copies/μL.IN conclusion,the TaqMan fluorescent quantitative RT-PCR method established in this study has strong specificity,good stability and high sensitivity,which provides a method for BNeV detection and molecular epidemiological investigation.