【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Mg²⁺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1.83×10⁰ copies/μL,其敏感度是PCR的1 000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。更多还原

【Abstract】 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 Mg²⁺ 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 1.83×10⁰ copies/μL,and its sensitivity was 1 000 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 100%.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.更多还原