Sensitive and rapid detection of Vibrio cholerae O139 by loop-mediated isothermal amplification / 中华微生物学和免疫学杂志

ABSTRACT

Objective To develop a loop-mediated isothermal amplification method for rapid diag-nosing Vibrio cholerae O139 in inspection department and small-scale laboratory. Methods Four primers (2 inner primer and 2 outer primer) for the LAMP test were designed by targeting the wbfR gene of Vibrio chol-erae O139, and the reaction condition and reaction system of LAMP were optimized. Thirty Vibrio cholerae O139, 13 Vibrio cholerae reference strains, 10 O1 biotype Vibrio cholera* and 32 other enterobacterias were analyzed and the LAMP results were determined by visual inspection or electrophoretie analysis . Results All of the Vibrio cholerae O139's amplification products were observed as green by visual inspection and had a ladder-like pattern on the gel, but O1 biotype Vibrio cholera* and other enterobacteria's products were dis-played as orange by visual examination and had no ladder-like pattern on the gel. In addition, the reaction time of the LAMP method was only 1.5 h and the detection limit of this assay was 63 CFU/reaction. Con-clnsion LAMP assay targeting the wbfR gene of Vibrio cholera* O139 is rapid, specific, and sensitive for the detection of Vibrio cholerae O139. This method not only reduced the dependence of complicated equip-ment but also had a potential for wider use in inspection department, small-scale laboratory, emergency mo-tor vehicles and field survey.