中国兽医科学2025，Vol.55Issue(1)：48-55,8.DOI:10.16656/j.issn.1673-4696.2025.0013
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
摘要
Abstract
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 visuali-sation 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 tem-perature,Mg2+concentration,the addition of Bst DNA polymerase and dNTPs,as well as the optimal concentra-tion ratios of internal and external primers were screened.After amplification,it could be visualised by turbidimet-ric method and the colour development reaction after the addition of HNB,SYBR Green Ⅰ,and calcium xantho-phyll dye.The method exhibited good specificity for C.perfringens after detection of 6 common intestinal bac-teria.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Key words
Clostridium perfringens/LAMP/visual inspection分类
畜牧业引用本文复制引用
樊月圆,王瑾艺,刘亚波,茶金龙,白卫兵,四朗玉珍,富国文..羊源产气荚膜梭菌LAMP检测方法的建立[J].中国兽医科学,2025,55(1):48-55,8.基金项目
西藏自治区十四五重大专项(XZ202101ZD0001N) （XZ202101ZD0001N）
