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| 环介导等温扩增法检测牛奶中结核分枝杆菌条件的优化 | |
| 引用本文： | 杨敏,王慧党,于潞,吴长新,张辉,曹旭东,陈创夫.环介导等温扩增法检测牛奶中结核分枝杆菌条件的优化[J].中国畜牧兽医,2016,43(7):1688-1693. |
| 作者姓名： | 杨敏 王慧党 于潞 吴长新 张辉 曹旭东 陈创夫 |
| 作者单位： | 1. 石河子大学生命科学学院, 石河子 832003; 2. 石河子大学动物科技学院, 石河子 832003; 3. 石河子大学医学院, 石河子 832002; 4. 新疆地方与民族高发病教育部重点实验室, 石河子 832002 |
| 基金项目： | "十二五"国家科技重大专项（2013ZX10003003-002）；国家自然科学基金（31060333） |
| 摘 要： | 为优化环介导等温扩增（loop-mediated isothermal amplification，LAMP）法检测牛奶中结核分枝杆菌的条件，以结核分枝杆菌高度保守的16S rRNA基因为靶基因设计3对特异性引物（F3-B3、FIP-BIP、FLP-BLP）进行试验。比较改良后的CATB/NaCl法、细菌基因组DNA提取试剂盒及热裂解法提取其DNA的效率，确定最佳的提取方法。用灭菌处理过的牛奶对结核分枝杆菌悬液进行倍比稀释以确定该检测方法的敏感度。以牛奶中常见的布鲁氏菌、大肠杆菌、金黄色葡萄球菌、李斯特菌、巴氏杆菌、沙门氏菌为对照，对该方法进行特异性测试。结果表明，改良后的CTAB/NaCl法对牛奶中结核分枝杆菌DNA的提取效果要优于其他两种方法。LAMP法检测结核分枝杆菌的灵敏度为3×100 CFU/mL，对人工阳性乳中结核分枝杆菌检测的灵敏度为3×101 CFU/mL。 |
| 关 键 词： | 环介导等温扩增 结核分枝杆菌 牛奶 |
| 收稿时间： | 2015-12-10 |
Study on Optimization of the Conditions of Detecting Mycobacterium tuberculosis in Milk by Technique of Loop-mediated Isothermal Amplification |
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| YANG Min,WANG Hui-dang,YU Lu,WU Chang-xin,Zhang Hui,CAO Xu-dong,CHEN Chuang-fu.Study on Optimization of the Conditions of Detecting Mycobacterium tuberculosis in Milk by Technique of Loop-mediated Isothermal Amplification[J].China Animal Husbandry & Veterinary Medicine,2016,43(7):1688-1693. | |
| Authors: | YANG Min WANG Hui-dang YU Lu WU Chang-xin Zhang Hui CAO Xu-dong CHEN Chuang-fu |
| Institution: | 1. College of Life Sciences, Shihezi University, Shihezi 832003, China; 2. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China; 3. School of Medicine, Shihezi University, Shihezi 832002, China; 4. Key Laboratory of Xinjiang Endemic and Ethnic Disease of Ministry of Education, Shihezi 832002, China |
| Abstract: | To optimize the loop-mediated isothermal amplification (LAMP) method to detect Mycobacterium tuberculosis in milk,the highly conservative 16S rRNA gene of Mycobacterium tuberculosis was selected as a target to design specific primers.Compared the modified method of CATB/NaCl,bacterial genome DNA extraction kit and thermal cracking method to extract the DNA of Mycobacterium tuberculosis,the best approach was chosen.Mycobacterium tuberculosis suspension liquid was diluted with sterilization milk to confirm the susceptibility of this assay.And then Brucella,Escherichia coli,Staphylococcus aureus,Listeria monocytogenes,Bacillus pasteurii and Salmonella were used for specificity detection.The results showed that the modified method of CATB/NaCl was better than the others.The sensitivity of LAMP was 3×100 CFU/mL,and the specificity was 100%.The sensitivity of detecting Mycobacterium tuberculosis in milk by LAMP was 3×101 CFU/mL. |
| Keywords: | LAMP Mycobacterium tuberculosis milk |
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