Development of two recombinase polymerase amplification (RPA) and lateral-flow dipstick (RPA-LFD) techniques for the rapid visual detection of Aeromonas salmonicida

Abstract

Aeromonas salmonicida is the pathogen of furunculosis, causing a septicaemic infection that influences both salmonid and non-salmonid fish. Early diagnosis of these contagions is essential for disease surveillance and prevention, so that the rapid and sensitive approach is required. Herein, recombinase polymerase amplification (RPA) assay and RPA with a lateral flow dipstick (RPA-LFD) were produced for testing A. salmonicida. The RPA and RPA-LFD primer sets were devised based on the conserved fragment sequence of vapA gene. Then, RPA and RPA-LFD reaction systems were established, and the reaction temperature and time were optimized. After optimization, the RPA method was capable to test A. salmonicida within 10 min and the RPA-LFD method can detect A. salmonicida in only 5 min. The RPA and RPA-LFD methods exhibited high specificity with no cross-reaction with others strains. To assess sensitivity, a partial vapA gene was cloned, and serial plasmid dilutions were created ranging from 1×106 to 1×10− 1 copies/µL. The detection limit of RPA was 1×102 copies/µL and the detection limit of RPA-LFD was 1 copies/µL. For spiked turbot tissue samples, the sensitivity detecting of A. salmonicida was 1.2×101 CFU/mL and 1.2 CFU/mL by RPA and RPA-LFD, respectively. Therefore, our RPA and RPA-LFD assays exhibited excellent specificity and sensitivity, which provided two simple, fast and dependable methods to conduct large-scale field investigations of A. salmonicida in resource-limited settings.