Fast, simple and highly specific molecular detection of Vibrio alginolyticus pathogenic strains using a visualized isothermal amplification method

Abstract Background: Vibrio alginolyticus is an important pathogen that has to be closely monitored and controlled in the mariculture industry for its strong pathogenicity, quick onset after infection and high mortality rate to aquatic animals. Fast, simple and specific methods are demanded for its on-site detection to effectively control its outbreaks and prevent economic losses. Detection specificity towards the pathogenic strains has to be emphasized to facilitate pointed treatment and prevention. Polymerase chain reaction (PCR) based molecular approaches have been developed, but their application is limited due to the requirement for complicated thermal cycling machines and trained personnel. Results: A fast, simple and highly specific detection method for V. alginolyticus pathogenic strains was established based on the isothermal recombinase polymerase amplification (RPA) and lateral flow dipsticks (LFD). The method targeted a virulence gene toxR that was reported to have a good coverage for the V. alginolyticus pathogenic strains. To ensure the specificity, the primer-probe set of the RPA system was carefully designed to recognize regions in gene toxR that were diverged in different Vibrio species but conserved in V. alginolyticus pathogenic strains. The primer-probe set was determined after a systematic screening on amplification performance, primer-dimer formation and false positive signals.The RPA-LFD method was confirmed to have a high specificity to V. alginolyticus pathogenic strains without any cross reaction with other Vibrio species, and was able to detect as low as 1 colony forming unit per microliter of the bacterium without DNA extraction. The method finishes detection within 30 min under the temperature between 35oC and 45oC, and the visual signal on the dipstick was directly read with naked eye. In an application simulation, randomly infected shrimp homogenate samples were 100% accurately detected. Conclusions: ​The RPA-LFD method developed in this study is fast, simple, highly specific and independent of complicated equipment. It is well applicable to the on-site detection of V. alginolyticus pathogenic strains for the mariculture industry.