【摘要】 为实现醋酸菌的快速灵敏检测,以醋化醋杆菌为代表菌株,首次使用环介导等温扩增技术(LAMP)进行16S-23S ITS r RNA核酸扩增,通过特异性检测后对反应条件进行优化,并对扩增产物的检测方法进行比较。结果表明,该组引物特异性强,优化的25μL体系为:6 mmol/L Mg Cl2,1.4 mmol/L d NTPs,内引物(FIP和BIP)各0.8μmol/L,外引物(F3和B3)各0.2μmol/L,1×Bst DNA聚合酶反应缓冲液,8U Bst DNA聚合酶,0.8 mmol/L甜菜碱,2μL DNA模板,用灭菌蒸馏水补足体系。62℃反应30 min即可完成扩增反应,产物经琼脂糖凝胶电泳后检测灵敏度达3.63×101CFU/m L。用肉眼观察白色焦磷酸镁沉淀,仅观察到3.63×102 CFU/m L以上的阳性产物,而在终产物中加入10 000×0.1μL SYBR GreenⅠ后,可通过肉眼观察产物颜色变化,得到与琼脂糖凝胶电泳法相同的检出限,并且缩短了检测时间。更多还原

【Abstract】 In order to develop a method for the rapid and specific detection of acetic acid bacteria, the Acetobacter actei was took as the model strain, the 16S-23 S r RNA nucleotide sequence was selected for primer design and Loopmediated isothermal amplification(LAMP) technology was used to test Acetobacter actei for the first time. The specificity of the primers was detected and the reaction system and reaction condition was optimized. Then the detecting methods for the products were compared. Results indicated LAMP primers had strong specificity, the 25 μL optimized reaction system contained 6 mmol/L Mg Cl2, 1.4 mmol/L d NTPs,0.8 μmol/L inner primers, 0.2 μmol/L outer primers, 1 ×Bst DNA polymerase thermal buffer, 8U Bst DNA polymerase, 0.8 mmol/L betaine, 2 μL DNA template, and sterilized distilled water.The reaction achieved the positive results in 30 minutes at 62 ℃ in the optimized reaction system, the detection sensitivity was up to 3.63×101CFU/m L which was examined by agarose gel electrophoresis. The white magnesium pyrophosphate precipitate visual inspection sensitivity was up to 3.63 ×102CFU/m L, and the naked eye detection limit by color change was achieved by adding 10 000× 0.1 μL SYBR Green Ⅰdye after reaction to the same detection sensitivity as agarose gel electrophoresis detection and the detection time was also shortened.更多还原