Xu Yigang, Li Sulong, Li Dandan, Zhang Hongxiang, Jiang Yanchun
Technical Centre of Heilongjiang Entry-exit Inspection and Quarantine Bureau, Harbin 150001, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Mar;26(3):398-403.
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 degrees 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.
霍乱弧菌是一种重要的食源性病原体，主要引起急性肠道传染病。开发快速检测霍乱弧菌的方法对于早期诊断其感染至关重要。在本研究中，根据霍乱弧菌管家基因mdh设计了两对特异性引物。经过反应优化，成功建立了用于快速检测霍乱弧菌的DNA环介导等温扩增（LAMP）方法。LAMP检测的最佳反应条件是65℃反应60分钟，培养的霍乱弧菌检测限为25 CFU/mL，其污染食品的检测限为32 CFU/g。使用33种同属或密切相关细菌确定该检测方法的特异性，只有霍乱弧菌菌株能被特异性扩增。在实际应用中，用LAMP方法从1057份虾、蟹、牡蛎、肉类和人类腹泻复合物样本中检测出85份阳性样本，与ISO TS 21872-1-2007的检测结果一致。因此，本研究建立的LAMP检测方法是一种检测霍乱弧菌的灵敏、快速且简便的工具，将有助于对其进行监测控制。
