兔源F型多杀性巴氏杆菌环介导等温扩增快速检测方法的建立  

Development of loop-mediated isothermal amplification assay forrapid detection of Pasteurella multocida serogroup F strain in rabbits

作　　者：王锦祥[1] 孙世坤[1] 陈岩峰[1] 陈冬金[1] 桑雷[1] 谢喜平[1] WANG Jinxiang;SUN Shikun;CHEN Yanfeng;CHEN Dongjin;SANG Lei;XIE Xiping(Institute of Animal Husbandry and Veterinary Medicine Fujian Academy of Agriculture Sciences,Fuzhou 350013 China)

机构地区：[1]福建省农业科学院畜牧兽医研究所,福建福州350013

出　　处：《中国兽医学报》2022年第3期490-495,共6页Chinese Journal of Veterinary Science

基　　金：福建省自然科学基金资助项目(2020J01346);福建省科技计划公益类专项资助项目(2020R10260015);国家现代农业产业技术体系资助项目(CARS-43-G-5)。

摘　　要：为了建立检测兔源F型多杀性巴氏杆菌的快速检测方法,本试验根据F型多杀性巴氏杆菌fcbD基因的保守序列设计了2对特异性引物,经反应条件优化,建立了检测兔源F型多杀性巴氏杆菌的环介导等温扩增(LAMP)快速检测方法。结果显示,该LAMP快速检测方法的最优反应条件为:外引物和内引物的浓度比为1∶7、反应温度为65℃、反应时间为60 min。该方法特异性强,对兔源A型和D型多杀性巴氏杆菌、支气管败血波氏杆菌、肺炎克雷伯菌、大肠杆菌、金黄色葡萄球菌和阴性对照(灭菌ddH_(2)O)均无交叉反应。该方法敏感性高,最低检出限为1×10^(2)拷贝/μL的兔源F型多杀性巴氏杆菌基因组DNA,是普通PCR方法的10倍。此外,该方法重复性好,准确性高。该方法的建立为兔源F型多杀性巴氏杆菌的快速检测提供了有力的技术支撑。In order to develop a rapid method for detection of Pasteurella multocida(P.multocida) serogroup F strain in rabbits, a loop-mediated isothermal amplification(LAMP) assay was established based on two sets of primers targeting the conserved sequences of fcbD gene of P.multocida serogroup F strain.The results showed that the optimal reaction conditions of the assay were as follows: the concentration ratio of external primer to internal primer was 1∶7,the reaction temperature was 65℃ and the reaction time was 60 min.The assay was specific for P.multocida serogroup F strain, and had no cross-reactions with P.multocida serogroup A and D strains, Bordetella bronchiseptica,Klebsiella pneumonia,Escherichia coli,Staphylococcus aureus.The detection limit of the assay was 1×10^(2)copies/μL of genomic DNA of P.multocida serogroup F strain, which was 10 fold higher than that of conventional duplex PCR assay.Moreover, the assay was repeatable and accurate.Taken together, the assay provides a strong technical support for the rapid detection of the P.multocida serogroup F strain in rabbits.

关 键 词：兔 F型多杀性巴氏杆菌 fcbD基因 LAMP快速检测方法 

分 类 号：S852.61[农业科学—基础兽医学]