Development of an <scp>RPA</scp> ‐ <scp>CRISPR</scp> /Cas12a‐Based Assay for Sensitive and Visual Detection of Channel Catfish Calicivirus ( <scp>ChCV</scp> )

ABSTRACT Channel catfish calicivirus (ChCV) has emerged as a novel viral pathogen threatening the sustainability of channel catfish ( Ictalurus punctatus ) aquaculture. The absence of rapid, on‐site detection methods has hindered effective disease prevention and control. In this study, a visual RPA‐CRISPR/Cas12a detection platform targeting the conserved non‐structural protein (NS) gene of ChCV was established, and the key reaction parameters were systematically optimized. The optimal combination was identified as the F3/R3 primer pair with crRNA3‐2. The optimal reaction conditions included RPA amplification at 37°C for 30 min, followed by CRISPR/Cas12a detection at 37°C for 30 min using 250 nM ssDNA reporter, 50 nM crRNA3‐2 and 200 nM Cas12a. Specificity analysis showed that the established system specifically recognizes ChCV without cross‐reactivity with other common aquatic pathogens affecting channel catfish, including channel catfish virus (CCV, also known as Ictalurid herpesvirus 1, IcHV1), channel catfish reovirus (CCRV), Edwardsiella tarda , Aeromonas hydrophila and Aeromonas veronii . Sensitivity evaluation demonstrated that the fluorescence visualization mode of the system achieved a limit of detection (LOD) of 2 copies/μL, while the lateral flow dipstick (LFD) mode exhibited an LOD of 2 × 10 2 copies/μL. Both detection modes were significantly more sensitive than conventional PCR. When applied to 20 clinical samples, the RPA‐CRISPR/Cas12a system yielded a positive detection rate of 60%, substantially higher than that of conventional PCR (25%). In conclusion, the RPA‐CRISPR/Cas12a system established in this study exhibits high specificity, ultra‐sensitivity and operational simplicity, making it a promising tool for rapid and field‐deplorable detection of ChCV.