Establishment of universal loop-mediated isothermal amplification method (LAMP) for rapid detection of pathogenic Vibrio spp. in aquatic organisms

Abstract

Pathogenic Vibrio spp. are regarded as the causative agent of prevalent vibriosis in aquatic organisms and food-borne zoonotic diseases of human, therefore, a rapid and effective detection method is useful for confirming early stages of the infection when the bacterial infection is at a relatively low level, which would significantly reduce the economic loss in aquaculture and seafood industry. Loop-mediated isothermal amplification (LAMP) is a convenient diagnostic method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a universal diagnostic protocol was successfully developed for the detection of Vibrio spp. in aquatic organisms. Based on 16S rRNA gene conservative sequence of genus Vibrio, a set of four primers was designed. The reaction time and temperature were optimized for 60 min at 62°C, respectively. The detection limit using the universal LAMP method was up to 103 cfu/ml and it was 100 times higher than the detection limit of the standard PCR method. The amplification products were detected by visual inspection using SYBR Green I. The specificity of the LAMP was evaluated by using 30 strains of nine Vibrio species and 21 related non-Vibrio micro-organism strains as controls. The target 30 Vibrio strains were all amplified, and no cross-reaction was found with all the non-Vibrio micro-organism strains. In conclusion, the universal LAMP assay should be a potential tool with high convenience, rapidity, sensitivity and specificity for the detection of Vibrio spp. in infected aquatic organisms, thereby facilitating surveillance of vibriosis. Key words: Loop-mediated isothermal amplification (LAMP), pathogenic Vibrio spp., detection, aquatic organisms, China