羊源产气荚膜梭菌LAMP检测方法的建立  

Establishment of a LAMP detection method for Clostridium perfringens in sheep

作　　者：樊月圆 王瑾艺 刘亚波 茶金龙 白卫兵 四朗玉珍[3] 富国文 FAN Yueyuan;WANG Jinyi;LIU Yabo;CHA Jinlong;BAI Weibing;SILANG Yuzhen;FU Guowen(College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650000,China;Yunnan Vocational and Technical College of A griculture,Kunming 650031,China;Institute of Animal Husbandry and Veterinary Medicine,Tibet Academy of Agricultural and Animal Husbandry Sciences,Lhasa 850000,China)

机构地区：[1]云南农业大学动物医学院,云南昆明650000 [2]云南农业职业技术学院,云南昆明650031 [3]西藏自治区农牧科学院畜牧兽医研究所,西藏拉萨850000

出　　处：《中国兽医科学》2025年第1期48-55,共8页Chinese Veterinary Science

基　　金：西藏自治区十四五重大专项(XZ202101ZD0001N)。

摘　　要：本研究旨在基于环介导等温扩增(LAMP)技术建立一种快速诊断羊源产气荚膜梭菌的可视化快速检测方法。首先以产气荚膜梭菌cpa基因为靶基因,构建重组质粒;建立LAMP检测技术并对各反应条件进行优化,随后开展敏感性、特异性、重复性试验,扩增产物可视化及临床样品检测。结果显示,本研究成功建立了羊源产气荚膜梭菌的LAMP检测方法,筛选出最适温度、Mg^(2+)浓度、Bst DNA聚合酶和dNTPs添加量以及内外引物最佳浓度比。并通过浊度法或添加HNB、SYBR GreenⅠ、钙黄绿素染料显色,实现可视化。使用该方法对6种常见肠道菌进行检测,仅对产气荚膜梭菌具有良好的特异性;以DNA为模板时,其灵敏度与荧光定量PCR法相近,是普通PCR的100倍;以菌液为模板时,其灵敏度比普通PCR法高出1~2个数量级,兼具便捷快速的特点。结果表明,本研究成功建立了羊源产气荚膜梭菌LAMP法,该方法可用于临床样品的快速检测。This study aimed to establish a visual rapid method for diagnosis of Clostridium perfringens of sheep origin based on the loop-mediated isothermal amplification(LAMP)technology.Initially,a recombinant plasmid was constructed targeting cpa gene of C.perfringens.After establishment of LAMP reaction system,the reaction conditions were optimized.Subsequently,the method for sensitivity,specificity,repeatability and visualisation were validated and clinical samples were performed using LAMP to assess it.The results showed that the LAMP detection method for C.perfringens of sheep origin was successfully established,and the optimal temperature,Mg^(2+)concentration,the addition of Bst DNA polymerase and dNTPs,as well as the optimal concentration ratios of internal and external primers were screened.After amplification,it could be visualised by turbidimetric method and the colour development reaction after the addition of HNB,SYBR Green Ⅰ,and calcium xanthophyll dye.The method exhibited good specificity for C.perfringens after detection of 6 common intestinal bacteria.The sensitivity of LAMP was similar to that of fluorescent PCR and is 100 times higher than that of ordinary PCR when DNA was used as template.Furthermore,its sensitivity was one to two orders of magnitude higher than ordinary PCR with convenience and rapidity when bacterial fluid was used as template.These results suggest that the LAMP method is suitable for the rapid detection of sheep-derived C.perfringens in clinical practice.

关 键 词：产气荚膜梭菌 环介导等温扩增技术 可视化检测 

分 类 号：S852.616.3[农业科学—基础兽医学]