伪结核棒状杆菌可视化LAMP现场检测方法的建立  

Development of visual loop-mediated isothermal amplification assay for field detection of Corynebacterium pseudotuberculosis

作　　者：李富祥[1] 夏九鲜 喻永福 朱沛 杨仕标[1] 赵文华[1] 高华峰[1] LI Fu-xiang;XIA Jiu-xian;YU Yong-fu;ZHU Pei;YANG Shi-biao;ZHAO Wen-hua;GAO Hua-feng(Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory,Yunnan Animal Science and Veterinary Institute,Kunming 650224,China;Agriculture and Rural Bureau of Xihan District,Kunming 650100,China;Animal Disease Prevention and Control Center of Shilin Yi Autonomous County,Shilin 652200,China)

机构地区：[1]云南省畜牧兽医科学院,云南省热带亚热带动物病毒病重点实验室,云南昆明650224 [2]昆明市西山区农业农村局,云南昆明650100 [3]石林彝族自治县动物疾病预防控制中心,云南石林652200

出　　处：《中国兽医科学》2023年第7期837-842,共6页Chinese Veterinary Science

基　　金：云南省重大科技专项计划资助项目(202102AE090039);云南省农业厅畜禽种质创新及标准化养殖技术推广专项资助项目。

摘　　要：为建立伪结核棒状杆菌可视化LAMP检测方法,根据伪结核棒状杆菌16S rRNA基因的特异性保守核苷酸序列设计2对引物,通过反应体系和反应条件的优化,建立了伪结核棒状杆菌可视化LAMP现场检测方法,并评价该方法的特异性和敏感性。结果显示,本实验所建立LAMP方法的最佳反应条件为62℃1 h。利用该方法检测大肠杆菌、肠炎沙门菌、金黄色葡萄球菌等山羊和绵羊常见的15种细菌的DNA样品,结果显示仅伪结核棒状杆菌检测为阳性,其他细菌检测为阴性。该方法对阳性质粒标准品pMD19-Cory的最低检出浓度为4.7 copies/μL。应用建立的LAMP方法和普通PCR方法对128份山羊皮下脓肿样品进行检测,结果显示LAMP方法和普通PCR方法的阳性率分别为21.1%(27/128)和20.3%(26/128),2种检测方法的总符合率为99.2%(127/128)。结果表明,本研究建立的可视化LAMP检测方法具有特异性强、敏感性高、快速的优点,为山羊和绵羊伪结核棒状杆菌感染的可视化现场快速诊断提供可行方法。To establish a visual LAMP method for the detection of Corynebacterium pseudotuberculosis(C.pseudotuberculosis),two pairs of primers were designed based on the specific conserved nucleotide sequence of the 16S rRNA gene of C.pseudotuberculosis,and the visual LAMP detection method was devel-oped by optimization of reaction system and reaction conditions,and it’s specificity and sensitivity were evaluated.The results showed that the optimal reaction condition of LAMP method was 62℃for 1 h.The method was used to detect DNA samples of 15 common bacterial species in goats and sheep,such as Es-cherichia coli,Salmonella enteritidis and Staphylococcus aureus,the results showed that only C.pseu-dotuberculosis was positive and the other bacterial species were negative.The minimum detected con-centration of positive plasmid standard pMD19-Cory was 4.7 copies/μL.One hundred and twenty-eight goat subcutaneous abscess samples were detected by the LAMP method and conventional PCR method,the re-sults showed that the positive rates in the LAMP method and PCR method were 21.1%(27/128)and 20.3%(26/128),respectively,and the total coincidence rate of the two methods was 99.2%(127/128).The results indicated that the established visualization LAMP was a specific,sensitive and rapid method,which provided a credible fast visual field diagnosis for C.pseudotuberculosis infections in goats and sheep.

关 键 词：伪结核棒状杆菌 环介等温扩增 可视化现场检测 

分 类 号：S852.618[农业科学—基础兽医学]