新城疫病毒强弱毒株LAMP鉴别检测方法的建立  

Development of a loop-mediated isothermal amplification assay for differentiation of Newcastle disease virus virulent strains from attenuated strains

作　　者：陈安莉[1] 谢芝勋[2] 周辰瑜[1] 刘加波[2] 庞耀珊[2] 邓显文[2] 谢志勤[2] 谢丽基[2] 

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]广西兽医研究所,广西南宁530001

出　　处：《中国兽医科学》2011年第9期917-922,共6页Chinese Veterinary Science

基　　金：国家百千万人才工程人选专项(945200603);广西科技攻关项目(桂科攻10100014-5);科技攻关与新产品试制项目(桂科攻1123007-4)

摘　　要：根据新城疫病毒(NDV)融合基因(F基因)的8个区域设计能区分强、弱毒株的3套特异性引物,以样品的cDNA为模板,利用Bst DNA聚合酶,在62℃恒温下进行扩增,扩增产物中加入SYBR GreenⅠ染料直接或在紫外光下观察判定结果,建立了能鉴别检测NDV强、弱毒株的环介导等温扩增方法。该方法可区别不同基因型的NDV强、弱毒株,而对常见鸡源病毒均无扩增反应。该方法对NDV RNA的最小检测限为0.01pg,灵敏度是常规RT-PCR方法的100倍。该方法无需特殊仪器,只需在常规水浴锅中进行,是一种适于基层的简便、灵敏、快速的NDV强、弱毒株鉴别检测方法。To develop a rapid and practical method for the differentiation of virulent strains of Newcastle disease virus（NDV） from the attenuated strains,a loop-mediated isothermal amplification（LAMP） assay was established with a set of primers based on the F fusion gene sequence of NDV.The viral cDNA generated by reverse transcription was amplified with Bst DNA polymerase at a constant temperature of 62 ℃,and products could be visualized under UV light with SYBR GreenⅠ dye.The LAMP assay established in the present study was specifically for the detection of different genotypes of NDV strains,but not for other avian respiratory pathogens.Detecting limit of the LAMP assay was 0.01 pg,which was 102-fold higher than that of conventional RT-PCR.The assay was therefore a sensitive,simple,rapid and practical method for the detection of NDV in the field.

关 键 词：新城疫病毒 强弱毒株 环介导等温核酸扩增 区分 

分 类 号：S852.659.6[农业科学—基础兽医学]