环介导等温扩增法检测牛奶中结核分枝杆菌条件的优化

摘要

To optimize the loop-mediated isothermal amplification (LAMP)method to detect My-cobacteriumtuberculosis in milk,the highly conservative 16S rRNA gene of Mycobacteriumtuber-culosis 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.%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16 S 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。