猪瘟兔化弱毒疫苗RT-LAMP检测方法的建立  

Development of a RT-LAMP method for detection of HCLV vaccine against classical swine fever

作　　者：张兴娟[1,2] 韩秋影[1] 孙元[1] 李宏宇[1] 常天明[1] 石火英[2] 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：《中国兽医科学》2010年第7期701-707,共7页Chinese Veterinary Science

基　　金：国家科技支撑计划项目(2006BAD06A03)

摘　　要：根据GenBank中登录的猪瘟病毒(CSFV)野毒株、猪瘟兔化弱毒疫苗(HCLV)和牛病毒性腹泻病毒(BVDV)的NS5B基因序列,利用在线软件Pri mer Explorer V4Software,设计了一套环介导逆转录等温核酸扩增(RT-LAMP)引物,建立了特异性检测HCLV的RT-LAMP方法。在BstDNA聚合酶作用下,65℃恒温反应1h即可完成扩增过程,扩增产物可通过20g/L琼脂糖凝胶电泳或加入SYBR GreenⅠ染料进行分析。经检测,该方法对于CSFV、BVDV以及其他猪源病毒无特异性扩增,具有良好的HCLV特异性;其灵敏度与本实验室建立的TaqMan实时荧光定量RT-PCR检测结果一致,均达到5个拷贝。分别用这两种方法对58份市售猪瘟弱毒疫苗进行检测,结果符合率达100%。表明,建立的HCLV RT-LAMP技术可以应用于基层实验室或养殖场,便于养殖户对疫苗中的弱毒含量进行监测。In order to develop a reverse transcription loop-mediated isothermal amplification（RT-LAMP）assay for the rapid detection of hog cholera lapinized virus（HCLV）vaccine strain against classical swine fever,a set of primers,being composed of outer primers and inner primers,was designed for NS5B gene of HCLV using Primer Explorer V4 software.After being amplified at a constant temperature of 65℃ by Bst DNA polymerase,a ladder-like pattern of products was visible on 20g/L agarose gel electrophoresis,and the products could be visualized under the UV light with SYBR GreenⅠ dye.The sensitivity of this assay was evaluated by comparison with the TaqMan real-time RT-PCR described previously.The detection limit of the assay was 5 copies of HCLV per reaction.No cross-reactivity was observed in the samples with other related viruses including different genotypes of wild-type CSFV strains and bovine viral diarrhea virus.The agreement between RT-LAMP and the TaqMan real-time RT-PCR was 100% in detecting 58 HCLV vaccines.The results indicated that the developed RT-LAMP was simple and rapid for the detection of HCLV.

关 键 词：猪瘟兔化弱毒疫苗 猪瘟病毒 反转录-环介导等温扩增 检测 

分 类 号：S852.659.6[农业科学—基础兽医学]