Establishment of Basic-RPA and RPA-LFD Rapid Detection Methods for Ranavirus in Largemouth Bass (Microterus salmoides).

Abstract

Largemouth bass ranavirus (LMBRaV) disease causes a high mortality rate in largemouth bass aquaculture industry. Establishment of rapid and sensitive virus detection assays is an urgency for prevention of virus transmission and disease outbreak. In this study, basic recombinase polymerase amplification (basic-RPA) and RPA combined with lateral flow dipstick (RPA-LFD) assays for LMBRaV were established to detect virus at low viral loads. The mcp gene of LMBRaV was the target sequence for primers and probes design. Then the primer concentrations, reaction temperature and time of basic-RPA and RPA-LFD assays were optimised. The basic-RPA was amplified under 38°C for 25 min, the results were detected by gel electrophoresis. The RPA-LFD assay required 15 min at 45°C for on-site visual results. The specificity of two assays showed that other aquatic viruses (CyHV-2, ISKNV, GSIV, WSSV and CrERV) could not be detected. The detection limit was 1 copy/μL DNA sample for both basic-RPA and RPA-LFD assays. Additionally, basic-RPA and RPA-LFD assays both detected nine more clinic samples compared to the PCR assay. Therefore, the basic-RPA as well as RPA-LFD assays provided more sensitive, rapid and simple operation methods for the on-site diagnosis of LMBRaV.