Development of two recombinase-aided amplification assays combined with lateral flow dipstick (RAA-LFD) and real-time fluorescence (RF-RAA) for the detection of Frog virus 3-like ranaviruses

Abstract

Frog virus 3-like ranaviruses (FV3-like viruses), particularly FV3 (Frog virus 3), represent typical species within the genus Ranavirus, primarily infecting amphibians and reptiles, thereby posing serious threats to aquaculture and biodiversity conservation. We designed a pair of universal primers and a probe targeting the conserved region of the major capsid protein (MCP) genes of FV3-like viruses. By integrating recombinase-aided amplification (RAA) with lateral flow dipstick (LFD) technology and real-time fluorescence (RF) modification, we established RAA-LFD and RF-RAA assays. The limit of detection (LoD) of RAA-LFD was determined to be 35.4 copies/μL under optimized amplification conditions at 35°C for 15 minutes. Similarly, the RF-RAA assay exhibited high specificity with a satisfactory LoD of 3.54 × 10² copies/μL at 39°C over a duration of 17-20 minutes. In diagnosing 53 clinical samples infected by an isolated strain of FV3-like viruses, both assays demonstrated consistency with results obtained from real-time fluorescence PCR assay. These experiments indicate that both methods serve as promising alternatives for point-of care testing (POST), adaptable to various scenarios. This study represents the first establishment of RAA-LFD and RF-RAA assays for FV3-likes viruses, enabling rapid and intuitive assessment of detection results while fulfilling on-site testing requirements, thus contributing positively to swift diagnosis and long-term monitoring in aquaculture.

Keywords: Frog virus 3-like ranaviruses (FV3-like viruses); lateral flow dipstick (LFD); point-of care testing (POST); real-time fluorescence (RF); recombinase-aided amplification (RAA).