LAMP方法检测霍乱弧菌的研究

Rapid detection of Vibrio cholerae by loop-mediated isothermal amplification method

目的建立一种快速检测霍乱弧菌的方法。方法利用霍乱弧菌的hlyA基因设计特异性引物,建立LAMP检测方法,以及优化反应体系,并进行LAMP的特异性和灵敏度实验,同时与普通PCR和荧光定量PCR进行比较。结果成功建立霍乱弧菌的LAMP检测方法,得到最优的反应体系,在特异性实验中,霍乱弧菌均呈阳性,而副溶血弧菌、溶藻弧菌、最小弧菌、1梅氏弧菌等均为阴性;在灵敏度实验中,霍乱弧菌的最低检测限为6.2pg/tube,是普通PCR的1000倍,比荧光定量PCR低一个数量级。结论LAMP方法具有很高的特异性和灵敏度,可用于霍乱弧菌的检测。

Objective To establish a specific,sensitive and rapid method for detecting Vibrio cholerae. Method hlyA genes was used for the design of primers, the reaction conditions were optimized and then LAMP method was established. Specificity and sensitivity evaluations also included in the experiment. Results LAMP method was established successfully, it was proved to be a high specific method which could detect all Vibrio cholerae. While results of detection of Vibrio parahaemolyticus , Vibrio alginolyticus, Vibrio mimicus, Vibrio metschnikovii,etc, were all negative .The detection limit of LAMP was 6.2pg/tube for Vibrio cholerae, about 100 times of that of conventional ventional PCR. Conclusion The established LAMP was a specific and sensitive method for detecting Vibrio cholerae and it can be used for Rapid detection of V ibrio cholerae.