鸭疫里默杆菌LAMP-LFD快速检测方法的建立与初步应用  

Establishment and preliminary application of LAMP-LFD rapid detection method for Riemerella anatipestifer

作　　者：龙宥茨 顾庆林 鲜思美[1,2] 周飘 梁倩 郑维豪 吴琴 LONG Youci;GU Qinglin;XIAN Simei;ZHOU Piao;LIANG Qian;ZHENG Weihao;WU Qin(College of Animal Science,Guizhou University,Guiyang 550025,China;Institute of Animal Disease Research of Guizhou,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病研究所,贵州贵阳550025

出　　处：《中国兽医学报》2023年第10期2050-2055,共6页Chinese Journal of Veterinary Science

基　　金：贵州省大学生创新创业训练资助项目(S202110657085);贵州省农业攻关资助项目[黔科合支撑(2016)2506号]。

摘　　要：根据鸭疫里默杆菌(Riemerella anatipestfer,RA)ompA基因设计4条环介导等温扩增(LAMP)特异性引物和1条带有异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的DNA探针,利用横向流动试纸条(LFD)检测扩增产物。通过对反应条件包括温度、时间、内、外引物浓度比、Bst DNA 2.0聚合酶浓度、dNTPs浓度及Mg^(2+)浓度等优化后,建立了RA的LAMP-LFD检测方法。结果显示:在64℃下运用LAMP扩增核酸到结果判读只需33 min;LAMP-LFD可特异性检测鸭疫里默杆菌,巴氏杆菌核酸、大肠杆菌核酸、沙门菌核酸、葡萄球菌核酸、鸭瘟病毒均未检测出;LAMP-LFD对pMD19-T-ompA最低检测浓度为1.83×10^(0)copies/μL,其敏感度是PCR的1000倍。利用PCR和LAMP-LFD检测22份疑似RA临床样本,两者检出符合率为100%。研究建立的LAMP-LFD检测方法能快速、特异地检测RA,可作为早期诊断和检测RA的有效手段。According to the ompA gene of Riemerella anatipestifer(RA),loop-mediated isothermal amplification(LAMP)specific primers and one DNA probe labeled with fluorescein isothiocyanate(FITC)were designed,and the amplification products were detected by lateral flow dipstick(LFD).By optimizing the reaction conditions,including temperature,time,internal and external primer concentration ratio,Bst DNA 2.0 polymerase concentration,dNTPs concentration and Mg^(2+) concentration,etc.,the LAMP-LFD detection method of RA was established.The results showed that it only took 33 min to amplify nucleic acids by LAMP at 64℃;LAMP-LFD could specifically detect RA,but nucleic acids of Escherichia coli,Salmonella enteriditis,Pasteurella multocida,Staphylococcus and duck plague virus were not detected;the minimum detection concentration of LAMP-LFD for pMD 19-T-ompA was 1.83×10^(0)copies/μL,and its sensitivity was 1000 times than that of PCR.PCR and LAMP-LFD were used to detect 22 clinical samples of suspected RA,and the detection rate of both was 1oo%.In conclusion,the LAMP-LFD detection method established can detect RA rapidly and specifically,and can be used as an effective means for early diagnosis and detection of RA.

关 键 词：鸭疫里默杆菌 环介导等温扩增 OmpA基因 横向流动试纸条 

分 类 号：S855.12[农业科学—临床兽医学]