Onsite and visual detection of ranavirus in largemouth bass (Micropterus salmoides) by an isothermal recombinase polymerase amplification method

Abstract

Largemouth bass ranavirus (LMBV) has caused mortality in largemouth bass and led to huge economic losses in the aquaculture industry. Here, we developed a novel, fast, and simple isothermal recombinase polymerase amplification assay (RPA) for LMBV detection with primers designed on the basis of a fragment of the major capsid protein gene. The reaction conditions were optimized and the RPA method was specific for LMBV, as the DNA of other four Iridoviridae viruses (Singapore grouper iridovirus, soft-shelled turtle iridovirus, tiger frog virus, and large yellow croaker iridovirus), white spot syndrome virus, grass carp reovirus, and healthy largemouth bass could not be amplified. The detection limit of LMBV-RPA was 89 copies/μL, which was comparable to the sensitivity determined by real-time PCR. Finally, the RPA method was validated to be a simple, convenient, rapid, and field diagnostic tool for LMBV detection using 10 clinical samples. In this paper, the RPA method is firstly applied to the diagnosis and monitoring of LMBV infection in the aquaculture industry of largemouth bass, which shows great prospects for on-site diagnostics of LMBV using nearly free instruments.