Development of a one-pot integrated rapid detection method for white spot syndrome virus based on RAA-CRISPR/Cas12a technology.

Pathogenic microorganisms, particularly white spot syndrome virus (WSSV), pose a major threat to global shrimp aquaculture, causing mass mortalities and substantial economic losses. To address the urgent need for rapid, accurate, and field-deployable detection methods, this study developed an innovative one-pot RAA-CRISPR/Cas12a assay. The platform integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technology using sucrose-mediated density gradient phase separation in a closed-tube format: sucrose acts as a density modifier to form distinct layers, spatially isolating RAA amplification reagents from CRISPR/Cas12a detection components to avoid cross-interference and enable sequential reactions without manual intervention. Under isothermal conditions at 37℃ for 60 min, the optimized assay achieves a limit of detection as low as 1 copy/μL, validated by both fluorescence and lateral flow dipstick (LFD) readouts. High specificity was confirmed by the absence of cross-reactivity with four other prevalent shrimp pathogens: infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (VpAHPND). Clinical validation with 30 field samples showed concordant results with the chinese national detection standard (GB/T 28630.2-2012). This novel nucleic acid detection platform combines highly sensitive, excellent specificity, and user-friendly visual interpretation, making it highly suitable for point-of-care testing and large-scale disease surveillance in shrimp aquaculture.