Development of a triplex loop-mediated isothermal amplification method for rapid on-site detection of three Vibrio species associated with fish diseases

Abstract

A triplex loop-mediated isothermal amplification (triLAMP) method was developed to detect three major fish pathogens, i.e., Vibrio harveyi, Vibrio anguillarum and Vibrio alginolyticus. Three sets of four species-specific primers were designed with one or two restriction enzyme sites contained in the inner primers of each set. TriLAMP is performed with a combination of all three sets of primers in a single reaction. A subsequent restriction enzyme analysis is used for discriminating the specific Vibrio species which caused the positive reaction. The specificity of this method was validated against a total of 32 strains representing 19 different bacterial species. The sensitivities of the triLAMP assays were 102 to 103 times higher than those of the traditional PCR assays. Via modified processes of sample preparation and visual inspection of the LAMP products with fluorescent staining, the triLAMP method allowed convenient and sensitive detection of V. harveyi, V. anguillarum and V. alginolyticus in the tissues of experimentally infected fish. These results indicate that the triLAMP method developed in this study is a useful tool for sensitive and rapid on-site detection of Vibrio infection in fish.