猪流行性腹泻病毒TaqMan荧光定量PCR检测方法的建立
Development of TaqMan Fluorescence Quantitative PCR for Detection of Porcine Epidemic Diarrhea Virus

基于猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)的N基因,建立了PEDV的TaqMan探针RT-PCR检测方法。以标准质粒绘制log10拷贝数与Ct之间标准曲线的线性相关系数值为0.996,检测灵敏度为100拷贝/μL,对传染性胃肠炎病毒、繁殖与呼吸障碍综合征病毒、伪狂犬病毒和圆环病毒2型等几种猪病毒的检测结果均为阴性,批内和批间的变异系数均小于1%,表明该方法具有良好的特异性、灵敏性和稳定性。对不同代次细胞培养上清中的病毒样品进行检测,结果表明该方法能够满足PEDV野毒株分离过程中的病毒定量检测需要。
Targeting the conservative N gene,a TaqMan probe-based fluorescence quantitative RT-PCR assay was developed for detecting porcine epidemic diarrhea virus.The correlation coefficient of standard curve of log10 copy number of plasmid versus Ct was 0.996 testified by a 10 fold successive dilution of recombinant plasmid.The detection limit was 100 copies/μL with no cross reaction with transmissible gastroenteritis virus,porcine reproductive and respiratory syndrome virus,pseudorabies virus and porcine circovirus 2.The variation coefficients of both intra and inter-batch were below 1%.These results indicated that this assay was highly specific,sensitive and reproductive.The established method was further used to detect virus samples at different tissue culture passage levels,the results confirmed that it could meet the quantitative detection in the course of isolating PEDV wild strains.