梅迪-维斯纳病毒实时荧光定量PCR检测方法的建立

Real-time quantitative RT-PCR assay for detection of Maedi-visna virus

为建立检测梅迪-维斯纳病毒(MVV)的TaqMan实时荧光定量PCR方法,本研究根据MVV核苷酸保守序列设计引物和探针。以梯度稀释的含有MVV目的扩增片段的重组质粒作为标准品,进行定量PCR反应。结果显示:5.0×105～5.0×101拷贝范围内定量PCR均有"S"型扩增曲线,检测灵敏度为50拷贝。对羊的其他病毒核酸均无扩增反应。本研究建立的实时定量PCR方法,灵敏度高,特异性好,在MVV的快速检测中具有良好的应用前景。

A real-time quantitative RT-PCR assay was developed for detection of Maedi-visna virus（MVV）.Primers and probes were designed based on the sequence of MVV by Primer Express 2.0.The recombinant 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 50 copies,with a dynamic range of detection between 5×105 copies to 5×101 copies.The primers and probe were specific for MVV and did not react with other virus,including Capripox virus,pseudorabies virus and Foot-mouth disease virus.The real-time RT-PCR assay described here with high sensitivity,specificity and accuracy is considered to be a powerful tool for the rapid detection and quantification of MVV.