快速检测兔支气管败血波氏杆菌环介导等温扩增(LAMP)方法的建立及应用  

Establishment and application of a rapid method for detection of rabbit Bordetella bronchiseptica by a loop-mediated isothermal amplification

作　　者：李科[1,2] 刘燕[1] 肖琛闻[1] 韦强[1] 鲍国连[1] 季权安[1] 

机构地区：[1]浙江省农业科学院畜牧兽医研究所,浙江杭州310021 [2]浙江师范大学化学与生命科学学院,浙江金华321004

出　　处：《中国兽医学报》2013年第7期1003-1006,1041,共5页Chinese Journal of Veterinary Science

基　　金：国家兔产业技术体系项目(CARS-44-C-2);浙江省重大农业项目(2011C12028);浙江省公益技术研究农业项目(2011C22013)

摘　　要：利用Primer ExplorerV4软件,针对兔波氏杆菌16SrRNA基因设计4条LAMP引物,优化反应条件,检测特异性、敏感性并对临床样品进行检测。结果显示,LAMP反应在62℃恒温扩增1h即可完成,操作简便,不需要PCR仪等复杂仪器,结果可经电泳检测及肉眼观察;具有良好的特异性;敏感性为普通PCR的100倍,最低可检测到1.65×10-6 mg/L的细菌基因组DNA。用建立LAMP检测方法对32份疑似兔支气管败血波氏杆菌(Bb)样品检测呈阳性,与PCR检测结果相符。该方法的建立为Bb的临床快速检测提供了新的思路。A loop-mediated isothermal amplification （LAMP） assay was developed for rapid,sensi- tive and specific detection for rabbit Bordetella bronchiseptica （Bb）. A set of four primers were designed based on the Bb 16S rRNA gene sequence using Primer ExplorerV4 software. Reaction conditions, detecting specificity and sensitivity were optimized with clinical samples. The results showed that the method could save time,operated easily,no need PCR instrument and other com- plex instruments compared with the traditional PCR method;the result could be obtained by agar- ose gel electrophoresis or even eyes. The sensitivity of the LAMP was 100 times higher than that of general PCR,and the minimum detection dose was 1.65 × 10-6 mg/L of RB50 DNA. The estab- lishment of LAMP provided a promise and alternative method for rapid detection with Bb.

关 键 词：兔支气管败血波氏杆菌 16S RRNA LAMP 

分 类 号：S852.61[农业科学—基础兽医学]