猪瘟病毒野毒株RT-LAMP可视化检测方法的建立  

Visualized detection of wild-type classical swine fever virus using RT-LAMP

作　　者：张兴娟[1,2] 孙元[1] 刘大飞[1] 仇华吉[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室猪传染病研究室,黑龙江哈尔滨150001 [2]扬州大学兽医学院,江苏扬州225009

出　　处：《中国预防兽医学报》2009年第11期864-868,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划(2006BAD06A03)

摘　　要：本研究旨在建立一种可视化检测猪瘟病毒(CSFV)野毒株的反转录-环介导等温扩增方法(RT-LAMP)。根据CSFV的NS5B基因序列设计一套RT-LAMP引物,以样品的cDNA为模板,利用BstDNA聚合酶,在62℃恒温条件下进行扩增,扩增产物中加入SYBR GreenⅠ染料直接或在紫外光下观察判定扩增结果。该方法可检测出不同基因型的CSFV野毒株,其检出极限为2.5TCID50的CSFV,与实时荧光定量RT-PCR方法的敏感性相当;特异性试验表明,该方法对猪瘟兔化弱毒疫苗株(HCLV)、牛病毒性腹泻病毒以及其它常见猪源病毒均无扩增反应;通过对126份不同样品进行检测比较,该方法与实时荧光定量RT-PCR检测方法的符合率达100%,与引物-探针能量转移PCR方法的符合率为98.4%。该方法无需特殊仪器,是一种适用于基层的快速、简便的CSFV野毒鉴别检测方法。To develop a rapid and practical method to differentiate wild-type strains of classical swine fever virus （CSFV） and the attenuated C-strain, a reverse transcription-loop-mediated isothermal amplification （RT-LAMP） assay was established with a set of primers based on NS5B gene sequence. The viral cDNA generated by reverse transcription was amplified with Bst DNA polymerase at a constant temperature of 62 ℃, and the products could be visualized under the UV light with SYBR Green I dye. The RT-LAMP was able to detect different genotypes of wild-type CSFV strains, but not for the C-strain, bovine viral diarrhea virus or other swine viruses. The detection limit of the RT-LAMP assay was 2.5 TCID50 of CSFV, comparable to the sensitivity of the real-time RT-PCR, The agreement rate between RT-LAMP and the real-time RT-PCR or the primer-probe energy transfer real-time PCR was 100 % or 98.4 % in detecting 126 samples. Thus, the assay is a rapid, sensitive, simple and practical method for the detection of wild-type CSFV in the field.

关 键 词：猪瘟病毒 野毒株 RT-LAMP 鉴别诊断 

分 类 号：S852.65[农业科学—基础兽医学]