Rapid RPA-CRISPR/Cas12a detection of Vibrio coralliilyticus for coral aquaculture disease surveillance

Coral aquaculture is expanding globally to support both the ornamental aquarium trade and reef-restoration initiatives, yet disease outbreaks remain a threat to production, causing potential substantial coral mortality and economic losses. Effective disease management requires early pathogen detection; however, conventional molecular diagnostics are labor-intensive and poorly suited to time-sensitive aquaculture operations. Here, we developed a rapid diagnostic assay for the coral pathogen Vibrio coralliilyticus by implementing recombinase polymerase amplification (RPA) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a-based fluorescent signaling for sensitive and specific detection. The assay was optimized and validated against DNA from seven V. coralliilyticus strains and seven non-target Vibrio species, demonstrating high specificity and a detection limit <10 target copies per reaction within 30 minutes following DNA extraction. Performance of the assay was further evaluated using water from flow-through aquaria containing healthy and diseased Acropora tersa colonies, and results independently validated using droplet digital PCR (ddPCR). Although RPA-CRISPR/Cas12a consistently detected V. coralliilyticus across the tanks holding both healthy and diseased corals, ddPCR indicated substantially higher pathogen abundance in diseased tanks, suggesting that pathogen load rather than the presence alone is a more reliable indicator of disease risk. Incorporating rapid semi-quantification of pathogen abundance in future assay development could therefore improve disease prediction and management. This study demonstrates the potential of CRISPR-based diagnostics as a fast, sensitive, and field deployable tool for routine coral aquaculture biosecurity, enabling earlier intervention and more effective mitigation of disease outbreaks.