小肠结肠炎耶尔森氏菌可视化LAMP检测方法的建立  

Establishment of visual LAMP method for detection of Yersinia enterocolitica

作　　者：徐云明[1] 朱孟玲[1] 杨剑波[1] 孙智远 卞蓉蓉 陈爱宁 郭丽娟 孙玉琴 王一伊 郑墁墁 仲思远 周弇扬 李叶辉 柳增善[3] 任洪林[3] XU Yun-ming;ZHU Meng-ling;YANG Jian-bo;SUN Zhi-yuan;BIAN Rong-rong;CHEN Ai-ning;GUO Li-juan;SUN Yu-qin;WANG Yi-yi;ZHENG Man-man;ZHONG Si-yuan;ZHOU Yan-yang;LI Ye-hui;LIU Zeng-shan;REN Hong-lin(Institute of Animal Husbandry and Veterinary Medicine,Jiangsu Vocational College of Agriculture and Forestry,Jurong 212400,China;Jurong Bureau of Agricultural and Rural Affairs,Jurong 212400,China;Institute of Zoonosis,Jilin University,Changchun 130062,China)

机构地区：[1]江苏农林职业技术学院畜牧兽医学院,江苏句容212400 [2]句容市农业农村局,江苏句容212400 [3]吉林大学人兽共患病研究所,吉林长春130062

出　　处：《中国兽医科学》2021年第3期281-288,共8页Chinese Veterinary Science

基　　金：吉林省科技发展计划项目(20200402059NC);江苏省自然科学研究面上项目(18KJB230001);江苏农林职业技术学院培育类项目(2019kj029,2016kj010)。

摘　　要：通过优化LAMP反应体系中镁离子、甜菜碱、dNTP等反应组成成分,而后进行特异性试验,并优化钙黄绿素与氯化锰的比例放入LAMP体系内,最后应用LAMP检测含有小肠结肠炎耶尔森氏菌(耶氏菌)的肉品,从而建立一种能够检测耶氏菌并通过肉眼观察和判断试验结果的LAMP方法。结果显示,优化的25μL反应体系如下:9 mmol/L Mg2+、1.8 mmol/L dNTPs、1.0 mol/L betaine、0.2μmol/L外引物、0.8μmol/L内引物、1μL Bst DNA polymerase(8 U/μL)、2.5μL 10×thermpol reaction buffer、228μmol/L钙黄绿素、324μmol/L氯化锰,反应温度65℃孵育60 min。LAMP检测耶氏菌基因组DNA的最低检测限是85.3×10^(-6)ng/μL,比文献PCR方法灵敏性高出2个数量级。LAMP方法对耶氏菌g DNA的最低检测限对应的基因拷贝数最低检测限是35,对应的CFU最低检测限是7×10^(2)CFU/m L。本试验建立的这种可视化的检测耶氏菌的LAMP方法,为耶氏菌的检测提供了一种新的参考。For detecting Yersinia enterocolitica simply and quickly,a detection method of Y.enterocolitica was establish used by LAMP.Components in the LAMP system such as Mg2+,betaine and d NTPs were optimized,and the detection method specificity was analyzed.And then,Yersinia enterocolitica in mocked meat was detected using the established LAMP method.Additionally,the concentration of calcein and manganese chloride added in the LAMP system was optimized,so as to observe LAMP detection results with naked eyes.The results were as follows:the LAMP reaction system including 9 mmol/L Mg2+,1.8 mmol/L d NTPs,1.0 mol/L betaine,0.2μmol/L outer primer,0.8μmol/L inner primer,1μL Bst DNA polymerase(8 U/μL),2.5μL 10×thermol reaction buffer,228μmol/L calcein,324μmol/L manganese chloride was carried out at 65℃for 60 min.The detection limit of the established LAMP method was 85.3×10^(-6) ng/μL for g DNA of Y.enterocolitica,which was more sensitive than the normal PCR method by 100 times,and 35 copies for the gene,and 7×10^(2) CFU/m L for the bacteria.In this study,the sensitive and visible LAMP provided a new choice in detection method of Y.enterocolitica.

关 键 词：小肠结肠炎耶尔森氏菌 LAMP 可视化检测 

分 类 号：S852.723[农业科学—基础兽医学]