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

摘要

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.