Establishment and application of a detection method for chinese rice-field eels rhabdovirus (CrERV) using the RPA-CRISPR/Cas12a System.

The Chinese rice-field eel rhabdovirus (CrERV) is an emerging pathogen that causes hemorrhagic disease in Chinese rice-field eels (Monopterus albus), leading to epidemic outbreaks, mass mortality, and considerable economic losses in aquaculture. Thus, the development of rapid and reliable diagnostic tools for on-site detection is urgently needed to address this issue. In this study, we established an RPA-CRISPR/Cas12a-based assay for CrERV detection, which exhibited superior sensitivity, specificity, and stability. The assay achieved a detection limit of 10¹ copies/µL. Specificity testing confirmed the absence of cross-reactivity with five other major aquatic viruses, including Grass carp reovirus (GCRV-II), Spring viraemia of carp virus (SVCV), Largemouth bass virus (LMBV), Cyprinid herpesvirus 2 (CyHV-2), and White spot syndrome virus (WSSV). Reproducibility analysis showed intra- and inter-assay coefficients of variation below 10%. Analysis of the 26 clinical samples showed that the RPA‑CRISPR/Cas12a assay achieved a higher positivity rate (23.08%, 6/26) compared to qRT‑PCR (15.38%, 4/26), providing preliminary evidence for its diagnostic potential in detecting CrERV. Collectively, these findings indicate that the RPA-CRISPR/Cas12a platform is a highly sensitive, specific, and user-friendly tool for rapid CrERV surveillance in aquaculture settings.