猪流感病毒实时荧光定量RT-PCR检测方法的建立

摘要

A real-time quantitative RT-PCR assay was developed for detection of Swine influenza virus (SIV). Primers and probe were designed based on NP gene of SIV by Primer Express 2. 0 software. The plasmid containing the target sequence was constructed to detect the sensitivity and prepare the standard curve. The real-time RT-PCR assay had a detection limit of 20 copies, with a dynamic range of detection between 2×108 to 2×102 copies/μL. The standard curve was prepared based on the linear relationship between the amount of plasmid RNA and cycle threshold (Ct). The method is specific for SIV, which failed to react with the classical swine fever virus, pseudorabies virus, foot and mouth disease virus and porcine reproductive and respiratory syndrome virus nucleic acid. The real-time RT-PCR assay that described with high sensitivity, specificity and accuracy is considered to be a powerful tool for the rapid detection and quantification of SIV.%本研究选取猪流感病毒(swine influenza virus,SIV)的NP基因序列设计引物和探针,建立了检测SIV的TaqMan实时荧光定量PCR方法.以梯度稀释的含有SIV目的扩增片段的质粒作为标准品,进行定量PCR反应以确定检测灵敏度.2.0×108至2.0× 102拷贝/μL 7个数量级的范围内定量PCR有“S”型扩增曲线,检测灵敏度为20个拷贝/μL.根据病毒拷贝数与Ct值的关系绘制了标准曲线.该方法具有特异性,对猪瘟病毒、伪狂犬病病毒、口蹄疫病毒和猪繁殖与呼吸综合征病毒核酸都没有扩增反应.本研究建立的实时定量PCR方法,灵敏度高、特异性好,可以进行定量分析,在猪流感的快速检测上具有重要意义.