胡兴义,张双翔,冯旭芳,周碧君,文明,程振涛,王伟,王开功

• 1:贵州大学动物科学学院
• 2:贵州省动物疫病研究室
• 3:贵州省动物疫病预防控制中心

摘要(Abstract)：

为了建立一种适用于猪轮状病毒(PoRV)的逆转录-环介导等温核酸扩增(RT-LAMP)的快速、灵敏检测方法。依据GenBank上登录的PoRV VP7基因保守序列,设计了6条特异性引物,通过对外引物与内引物浓度比、Bst DNA聚合酶浓度、Mg2+浓度、dNTP浓度和反应条件等进行优化。结果显示,当外引物与内引物浓度比为200nmol/L∶2 400nmol/L(1∶12)、Bst DNA聚合酶浓度为0.64 U/μL、Mg2+浓度为2.5 mmol/L、dNTP浓度为1.0mmol/L,在恒温(60℃)条件下作用60min,扩增效果出现明显"梯状"条带,同时对建立的RT-LAMP检测方法进行特异性和敏感性验证,其只有PoRV获得特异性扩增条带,与其他猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪瘟病毒等无交叉反应,具有良好的特异性,最低检测分子拷贝数为1.0×102拷贝/μL,具有极高的敏感性。反应结束后肉眼可见阳性扩增产物出现白色沉淀,加入SYBR GreenⅠ观察颜色变化可以判定结果。该方法适用于野外、基层部门和海关快速检测PoRV的新方法,在临床上有良好的推广意义。

关键词(KeyWords)： 猪轮状病毒(PoRV);RT-LAMP;建立;应用

Abstract:

Keywords:

基金项目(Foundation): 贵州省科学技术厅农业攻关项目(黔科合NY[2015]3009-1号);; 贵州省生猪质量安全工程技术研究中心建设项目(黔科合农C字[2011]4022号);; “贵州省动物疫病防控与兽医公共卫生保障科技创新人才团队”(黔科合人才团队[2015]4016号)

作者(Author): 胡兴义,张双翔,冯旭芳,周碧君,文明,程振涛,王伟,王开功

DOI: 10.13242/j.cnki.bingduxuebao.003071

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