环介导等温扩增技术与横向流动试纸条法快速检测布鲁氏杆菌  

Rapid and Sensitive Detection of Brucella spp. by Loop-Mediated Isothermal Amplification Combined with a Lateral-Flow Dipstick

作　　者：李秀梅[1] 梁智选[1] 李颖[1] 郭恋[1] 王健春[1] 赵静[1] 王红军 LIXiumei;LIANG Zhixuan;LI Ying;GUO Lian;WANG Jianchun;ZHAO Jing;WANG Hongjun(Animal Disease Prevention and Control Center of Tianjin,Tianjin 300402,China;Animal Husbandry Development and Service Center of Tianjin,Tianjin 300384, China)

机构地区：[1]天津市动物疫病预防控制中心,天津300402 [2]天津市畜牧业发展服务中心,天津300384

出　　处：《食品与生物技术学报》2017年第12期1276-1282,共7页Journal of Food Science and Biotechnology

基　　金：国家863计划项目(2012AA101605)

摘　　要：将环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)与横向流动试纸条技术(Lateral flow dipstick,LFD)联合应用,建立了一种快速、便捷的布鲁氏杆菌检测方法。针对布鲁氏杆菌OMP25基因设计4条特异性引物和1条异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记的探针。生物素标记LAMP扩增产物能够特异性地与FITC标记的探针杂交,杂交产物经LFD检测。优化后的扩增温度和时间为63℃反应50 min,加上样品的前处理,完成检测仅需80 min。LAMP-LFD方法可特异性地检出布鲁氏杆菌,其最低检测限为5.4×100CFU/mL,是利用外引物建立的PCR方法的100倍。试验结果表明,本研究建立的LAMP-LFD方法能够快速、特异地检测布鲁氏杆菌;简单经济,不需要其他辅助仪器,结果即可直观判断;适合基层实验室、应急检测或现场监测等使用,具有较高的推广价值。In this study,we established a rapid Brucella spp. detection method by combining loop-mediated isothermal amplification （LAMP） and lateral flow dipstick （LFD） examination. The method used four specific primers targeting to Brucella spp. 0 MP25 gene for LAMP and one fluorescein isothiocyanate （FITC）-labeled probe to specifically hybrid to the biotin-labeled LAMP product for LFD examination. The optimized amplification temperature and time were 63 ! and 50 min, respectively and the total procedure time was only 80 min by taking sample process into account. The established LAMP-LFD method could specifically detect Brucella spp.. Its detection sensitivity for pure bacterial culture reached 5.4 xlO° CFU/mL, 100-fold lower than that of conventional PCR established using the outer primer set. Overall, the established LAMP-LFD method could rapidly and specifically detect Brucella spp. and is simple, economical and intuitive. In addition, it requires no other auxiliary equipment, thus it is suitable for basic laboratory tests, emergency use and in on-site monitoring and has high promotion value.

关 键 词：布鲁氏杆菌 OMP25基因 环介导等温扩增 横向流动试纸条 检测 

分 类 号：S852.61[农业科学—基础兽医学]