A rapid RPA-CRISPR/Cas12a approach for detecting red spotted grouper nervous necrosis virus

Red spotted grouper nervous necrosis virus (RGNNV) is one of the most significant pathogens threatening marine fish farming in China, so early detection of RGNNV is a critical measure for preventing viral infection and reducing aquaculture losses. To develop a rapid RGNNV detection method, we developed a rapid visual detection method by integrating recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system. Besides, we designed specific RPA primers and crRNA, then optimized both RPA reaction conditions and CRISPR/Cas12a detection parameters. The method's sensitivity and specificity were systematically evaluated through clinical testing of 20 suspected-infected grouper samples, with comparison to the standardized qPCR method (SC/T 7216—2022). Results show a detection limit of 1.5×10¹ copies·µL⁻¹ and complete concordance with qPCR findings. The established RPA-CRISPR/Cas12a method demonstrates rapid detection, high sensitivity, excellent specificity, and visual interpretability, providing an efficient field-deployable solution for RGNNV surveillance in aquaculture operation.