Development of a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay for the rapid on-the-spot detection of turbot circovirus

Since 2019, a novel condition termed ‘emerging acute hemorrhagic syndrome’ (EAHS) has been associated with significant mortality in farmed Scophthalmus maximus L. (turbot) populations across China. Turbot circovirus (TurCV) has been identified as a primary pathogen associated with this syndrome, contributing to high mortality rates and severe economic impacts in intensive aquaculture operations. To address these challenges, the establishment of an early, rapid and reliable diagnostic approach is essential for effective EAHS management in turbot farming. In this study, we developed and validated a real-time recombinase polymerase amplification (RT-RPA) assay, alongside RPA combined with lateral flow dipsticks (RPA-LFD) and agarose gel electrophoresis (RPA-AGE), targeting the conserved region of the TurCV Capsid gene. Both methods enable visual detection within 15 min at a constant temperature ranging from 25 to 45 °C. Using a synthetic plasmid as a template, the RPA-LFD assay demonstrated a detection limit of 10 3 copies/μl. When evaluated with DNA extracted from EAHS-affected turbot, both assays exhibited an identical detection limit of 10 3 copies/μl. Specificity testing confirmed the absence of cross-reactivity with other common aquatic pathogens, including turbot reddish body iridovirus (TRBIV), Scophthalmus maximus reovirus (SMReV), hirame rhabdovirus (HRV), viral hemorrhagic septicemia virus (VHSV), lymphocystis disease virus (LCDV), Edwardsiella piscicida , Aeromonas salmonicida , Vibrio alginolyticus , Vibrio anguillarum , Pseudomonas plecoglossicida or DNA from healthy turbot. Field evaluations using clinical samples corroborated the assays' practical utility, with results aligning with conventional PCR and qPCR. Analysis of clinical specimens revealed that both assays accurately distinguished between positive and negative cases, with the RPA assay achieving a TurCV detection rate of 93.00% in clinical samples and a 97.07% concordance rate with conventional PCR. In conclusion, the RPA-LFD assay represents a simple, rapid, sensitive and cost-effective diagnostic tool for TurCV detection, offering significant promise for controlling EAHS outbreaks and reducing economic losses in turbot aquaculture.