改良LAMP快速检测酸土脂环酸芽胞杆菌的研究

Development of Loop-mediated Isothermal Amplification Method for Rapid Detection of Alicyclobacillus acidoterrestris

建立改良环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测酸土脂环酸芽胞杆菌的方法。以酸土脂环酸芽胞杆菌的16S r RNA基因保守区域设计4条特异性引物,优化反应体系,通过荧光曲线、琼脂糖凝胶电泳和白色沉淀判定扩增结果。同时,对LAMP引物特异性、灵敏度、检出限进行研究。LAMP法在61℃,60 min内完成酸土脂环酸芽胞杆菌的检测。2株酸土脂环酸芽胞杆菌呈阳性结果,17株非酸土脂环酸芽胞杆菌呈阴性结果,表明该种检测方法具有高特异性。检测纯菌灵敏度为7.2 CFU/m L,对人工污染苹果汁样品中酸土脂环酸芽胞杆菌的检出限是18 CFU/m L。LAMP法检测酸土脂环酸芽胞杆有操作简单、灵敏度高、特异性强等优点,应用前景广阔。

Development of loop-mediated isothermal amplification method was established to detect for Alicyclobacillus acidoterrestris(A. acidoterrestris).Taking A. acidoterrestris 16 S rRNA gene specific sequence as the target sequence,the primer sequence was designed. The reaction conditions were optimized and results observed by fluorescence curve,agarose gel electrophoresis and turbidity. The specificity,sensitivity and detection limit were detected by this method.The detection could be complished at 61 °C within 60 min by LAMP. The specificity of the assay was confirmed using 2 A. acidoterrestris strains and 17 non-A. acidoterrestris strains. The detection sensitivity and detection limit were 7.2 CFU/m L,18 CFU/m L,respectively.The advantages of simple operation,high specificity and sensitivity of LAMP maked it to apply in future.