快速检测小反刍兽疫病毒RT-LAMP方法的建立  

Establishment of a rapid method for detection of peste des petits ruminants virus by a reverse transcription loop-mediated isothermal amplification

作　　者：李伟[1] 李刚[1,2] 范晓娟[1] 张坤[1] 贾风琴[1] 史利军[1] Hermann Unger 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]动物营养学国家重点实验室,北京100094 [3]FAO/IAEA农业生物技术实验室,Seibersdorf奥地利

出　　处：《中国预防兽医学报》2009年第5期374-378,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：“十一五”国家科技支撑计划(2006BAD06A13-4);国家“863”计划(2008AA10Z411);FAO/IAEA项目(14515-0,R1);“948”项目(2007-G57-C)

摘　　要：本研究采用一步法反转录环介导等温扩增技术(RT-LAMP)建立了一种快速、灵敏、高度特异的检测小反刍兽疫病毒(PPRV)的方法。针对PPRV-N基因保守区域设计4条引物,在Bst大片段聚合酶的作用下,可以实现DNA的梯状等温扩增。优化RT-LAMP的反应体系,并检验其灵敏性、特异性。一步法RT-LAMP检测方法从核酸抽提到检测结果出现仅需70 min,该方法具有良好的特异性,与同属的犬瘟热病毒无交叉反应,灵敏度是RT-PCR的1 000倍,是巢式RT-PCR的100倍。结果证明,RT-LAMP方法可快速、灵敏、特异、经济地检测PPRV,在基层和实验室都具有良好的应用前景。A one-step reverse transcription loop-mediated isothermal amplification （RT-LAMP） assay was developed for rapid and sensitive detection of peste des petits ruminants virus （PPRV）. A set of four primers were designed based on the PPRV N gene sequence; RT-LAMP was performed using Bst DNA polymerase large fragment, and ladder like DNA fragments were observed on agarose gel electrophoresis assay. The process takes only 70 minutes and the amplification results could be visualized with SYBR Green I. The sensitivity test indicated that the RT-LAMP was 1000 times higher than that of RT-PCR, while 100 times higher of nest RT-PCR. The method described in this study was proved to be a sensitive, specific and rapid way for the detection of PPRV in infected cells and tissues. It has a potential application of both laboratorial and pen-side detection of PPRV.

关 键 词：小反刍兽疫病毒 RT—LAMP 核酸检测 

分 类 号：S852.659.5[农业科学—基础兽医学]