牛溶血性曼氏杆菌可视化LAMP检测方法的建立  

Establishment of visual LAMP method for detecting Mannheimia haemolytica in cattle

作　　者：刘宁宁 易萍 郭爱珍[1,2,3] 胡长敏 陈颖钰[1,2] LIU Ningning;YI Ping;GUO Aizhen;HU Changmin;CHEN Yingyu(College of Veterinary Medicine,Huazhong Agricultural University/State Key Laboratory of Agricultural Microbiology,Wuhan 430070,China;Hubei International Scientific and Technological Cooperation Base of Veterinary Epidemiology(Huazhong Agricultural University),Wuhan 430070,China;Hubei Hongshan Laboratory,Wuhan 430070,China)

机构地区：[1]华中农业大学动物医学院/农业微生物资源发掘与利用全国重点实验室,武汉430070 [2]湖北省兽医流行病学国际科技合作基地,武汉430070 [3]湖北洪山实验室,武汉430070

出　　处：《华中农业大学学报》2023年第2期32-37,共6页Journal of Huazhong Agricultural University

基　　金：湖北省重点研发计划项目(2020BBA055);宁夏回族自治区重点研发计划项目(2021BEF02028);国家现代农业(肉牛/牦牛)产业技术体系专项(CARS-37)。

摘　　要：为建立一种能够快速、高效检测牛溶血性曼氏杆菌(Mannheimia haemolytica)的可视化LAMP检测法,利用LAMP引物设计软件,以溶血性曼氏杆菌保守基因lktC序列设计内引物、外引物及环引物,对其反应温度、Mg^(2+)浓度、dNTPs浓度等条件进行优化,并检测了方法的敏感性、特异性及实用性。结果显示,该方法在68.2℃、6 mmol/L Mg^(2+)、1.6 mmol/L dNTPs条件下达到最佳,40 min内完成检测并可通过肉眼判定结果,对细菌的最低检测限为6.7×10^(-1)cfu/μL,对lktC重组质粒的最低检测限为7.9×10^(2)copies/μL,敏感性高;与15种病原均不发生交叉反应,特异性好;对背景清晰小鼠肺组织样本检测结果显示该方法实用性良好,且优于普通PCR方法。表明该方法可快速、高效检测牛溶血性曼氏杆菌。In order to establish a quick and efficient visual LAMP(loop-mediated isothermal amplifi⁃cation)molecular method for detecting Mannheimia haemolytica in cattle,specific software tool was used to design LAMP primers including inner primers,external primers and loop primers targeting M.haemolyt⁃ica conservative gene lktC.The reaction temperature,Mg^(2+) concentration,dNTPs concentration and other conditions were optimized.Both bacteria and recombinant plasmids were used to evaluate sensitivity,and 15 related pathogens were used to evaluate specificity.The applicability was evaluated using mice lung tis⁃sue samples with clear background.The results showed that the developed LAMP assay achieved the best results under the conditions of 68.2℃,6 mmol/L Mg^(2+),1.6 mmol/L dNTPs for 40 min and could be judged by naked eyes.The minimum detection limits were 6.7×10^(-1) CFU/μL for bacteria and 7.9×10^(2) copies/μL for lktC recombinant plasmid,and there was no cross reaction with 15 other pathogens,indicat⁃ing high sensitivity and specify.The results of lung tissue samples from mice with clear background showed that the method was better than ordinary PCR method.The above results indicate that this method was suc⁃cessfully constructed and could be used for rapid and efficient detection of M.haemolytica in cattle.

关 键 词：环介导等温扩增(LAMP) lktC基因 溶血性曼氏杆菌 可视化检测 综合防控 

分 类 号：S852.61[农业科学—基础兽医学]