DNA环介导恒温扩增技术快速检测霍乱弧菌

Rapid detection of Vibrio cholerae by loop mediated isothermal amplification (LAMP) method

摘要:

霍乱弧菌是一种重要的食源性致病菌，主要引起急性肠道传染病，其快速检测具有重要意义。根据霍乱弧菌的mdh管家基因序列，设计2对特异性检测引物，利用DNA环介导恒温扩增技术 (Loop-mediated isothermal amplification，LAMP)，经反应体系优化，成功建立了霍乱弧菌的LAMP快速检测方法。该方法最佳反应温度为65℃，60 min完成检测，对培养菌的检测限为25 CFU/mL，污染食品中霍乱弧菌的检测限为32 CFU/g。对33株同种或近源细菌进行LAMP检测，仅霍乱弧菌得到阳性扩增。LAMP方法实践应用结果表明，对1057份虾、蟹、牡蛎、肉类、人腹泻物等样本进行检测，共检出85份阳性，与国际标准 (ISO TS 21872-1-2007) 检测结果的符合率为100％。结果表明，本研究建立的霍乱弧菌LAMP检测方法特异性强、灵敏度高、操作简便，有利于霍乱弧菌疫情的监测。

Abstract:

Vibrio cholerae is an important foodborne pathogen, mainly causes acute intestinal infectious disease. The development of rapid method for detecting Vibrio cholerae is critical for early diagnosis of its infection. In this study, two pairs of specific primers were designed according to housekeeping gene mdh of Vibrio cholerae. Following optimization of the reaction, DNA loop-mediated isothermal amplification (LAMP) for rapidly detecting Vibrio cholerae was successfully established. The optimal reaction for the LAMP assay is 65?C for 60 min, with detection limit for cultivated Vibrio cholerae of 25 CFU/mL and for its contaminated food of 32 CFU/g. The specificity of the assay was determined using thirty-three kinds of same species or closely related bacteria, only Vibrio cholerae strains were specifically amplified. In practice, 85 pieces of positive samples were detected from 1057 pieces of shrimps, crabs, oysters, meat and human diarrhea complex using the LAMP method, which accorded with the detection result by ISO TS 21872-1-2007. Thus, the LAMP assay established in this study is a sensitive, rapid and simple tool for detecting Vibrio cholerae and will facilitate the surveillance for its control.