禽新型黄病毒RT-LAMP检测方法的建立  

Establishment of Rapid Reverse Transcription Loop Mediated Isothermal Amplification for Detection of Avian Novel Flavivirus

作　　者：李兆龙[1,2] 陈仕龙[1,2] 林锋强[1,2] 王劭[1,2] 程晓霞[1,2] 朱小丽[1,2] 陈少莺[1,2] 

机构地区：[1]福建省农业科学院畜牧兽医研究所,福州350013 [2]福建省畜禽疫病防治工程技术研究中心,福州350013

出　　处：《畜牧兽医学报》2012年第4期659-663,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：福建省农科院创新团队项目(STIF-Y02);福建省公益类科研院所专项(2011R1025-2)

摘　　要：根据环介导等温扩增技术(LAMP),建立了一种适用于禽新型黄病毒的逆转录环介导等温扩增(RT-LAMP)快速检测方法。根据禽新型黄病毒E蛋白基因序列,在保守区设计了一套针对禽新型黄病毒基因8个区域的6条特异性引物,并对反应条件进行优化。结果表明该方法对NDRV、GPV、MDRV、NDV、ILTV、FPV均无扩增反应,并可通过反应液是否有沉淀或向反应液中加入荧光染料来对结果进行可视化观察;扩增反应只需要在常规水浴锅中进行,45min内可完成反应;该方法对禽新型黄病毒RNA的最小检测限为1pg,灵敏度是一步法RT-PCR方法的100倍。本研究建立的RT-LAMP方法简便、快速、灵敏、特异,适合在基层进行禽新型黄病毒的快速检测。A reverse transcription loop mediated isothermal amplification（RT-LAMP） assay was developed for detection of avian novel flavivirus virus.According to the sequences of avian novel flavivirus virus E protein gene,six primers specific to the eight sites of novel flavivirus virus gene were designed,and the reaction conditions were optimized.The results showed that the assay had no cross reaction with NDRV,GPV,MDRV,NDV,ILTV and FPV.The assay was performed in water bath within 45 minutes and the amplification result was visualized by adding SYBR Green Ⅰ or inspecting the white sediment.The detection limit of RT-LAMP assay was 1 pg,which was 100 fold higher than that of one step RT-PCR.The detection in clinical samples was 100% compliance with RT-PCR.These results suggested that the newly developed RT-LAMP assay is a simple and specific method for rapid detection of avian novel flavivirus virus in field conditions.

关 键 词：禽新型黄病毒 RT-LAMP 检测方法 

分 类 号：S852.659.6[农业科学—基础兽医学]