建立DARQ-LAMP方法快速检测单增李斯特菌

Development of DARQ-LAMP for Rapid Detection of Listeria monocytogenes

该研究针对单增李斯特氏菌的特异性基因hlyA设计引物,优化反应条件,建立实时荧光环介导等温扩增(realtime loop-mediated isothermal amplification,real-time LAMP)的检测方法,在此基础上,建立检测单增李斯特氏菌的基于淬灭基团释放环介导等温扩增检测方法(detection of amplification by release of quenching-LAMP,DARQ-LAMP),分析其特异性和灵敏度。结果表明,该方法的适宜反应条件为反应温度63℃、Bst DNA 3.0聚合酶0.32 U/μL、淬灭探针双链(quenching probe double chain,QPD)探针浓度10%、Mg2+浓度6 mmol/L;对单增李斯特菌的检测限为7.3×101copies/mL,灵敏度是普通聚合酶链式反应(polymerase chain reaction,PCR)的100倍。该方法效率高、特异性好、灵敏度高,为环介导等温扩增技术检测食源性致病菌的研究提供参考。

A real-time loop-mediated isothermal amplification(LAMP)assay was established to detect Listeria monocytogenes. The primers were designed for the specific gene hlyA of L. monocytogenes,and the reaction conditions were optimized. On this basis,an assay named detection of amplification by release of quenching-LAMP(DARQ-LAMP)was established,and its specificity and sensitivity were analyzed. The results showed that the optimum reaction conditions were 63 ℃,Bst DNA 3.0 polymerase of 0.32 U/μL,quenching probe double chain(QPD)concentration of 10%,and Mg2+concentration of 6 mmol/L. The established assay showed the limit of detection of 7.3 ×101copies/mL and the sensitivity 100 times that of the conventional polymerase chain reaction(PCR). This method exhibited high efficiency,specificity,and sensitivity for the detection of L. monocytogenes,providing a reference for the development of LAMP-based technology for rapid detection of food-borne pathogens.