羊梅迪-维斯那病毒实时荧光定量 PCR 检测方法的建立

摘要

为了建立一种能快速检羊梅迪-维斯那病毒（MVV）的实时荧光定量 PCR，根据 GenBank中MVV gag 基因的序列，设计了1对特异性引物及 TaqMan 探针，经过反应体系及条件优化，以定量的10倍系列稀释的阳性质粒为标准品进行实时荧光定量 PCR 扩增，建立了 MVV 实时荧光定量 PCR。结果显示，该方法对 MVV DNA 最小检出量为10 copies／μL，比普通 PCR 具有较高的检出率；组内及组间变异系数均低于2％，具有较好的重复性；该方法可以特异地检测到 MVV，与其他病毒性样品无交叉反应。被检的92份临床样品中，实时荧光定量 PCR 和常规 PCR 的阳性检出率分别为4．3％和3．3％。为羊 MVV 快速检测和分子流行病学调查提供了一种有效的方法。%In order to establish a TaqMan real-time PCR method to quickly check the sheep Maedi-Visna vi-rus (MVV),according to the MVV gag gene sequences in GenBank,a pair of specific primers and a Taq Man probe were designed after optimizing the reaction system and conditions.The standards which are pos-itive plasmids of quantitative 10-fold serial dilutions to real-time PCR amplification were used,and the real-time PCR detection method of MVV was established.The test results showed that the minimum detectable amount of this method is 10 copies,compared with ordinary PCR,the method has a higher detection rate;the coefficient of variation is less than 2% in intergroup and intragroup assays,with good repeatability;the method can be specific for detecting MVV,and do not have cross-reaction with other viral samples.Among 50 clinical samples,the positive rate was 4.3% detected by real-time PCR and 3.3% by conventional PCR. It indicated that the real-time PCR assay could be used for the rapid detection of MVV and molecular epide-miology investigation in sheep.