Field-Deployable Paper-Based Detection of <i>Vibrio vulnificus</i> via Mechanically Automated Sequential RPA/CRISPR-Cas12a

Vibrio vulnificus (V. vulnificus) is a highly virulent marine pathogen that causes severe infections, including sepsis and necrotizing fasciitis, with high fatality rates in susceptible individuals. Rapid and sensitive on-site detection is therefore essential, yet existing methods often depend on laboratory-based equipment and trained personnel, limiting field applicability. Here, we present an automated, paper-based RPA/CRISPR-Cas12a diagnostic platform that integrates a microfluidic paper pad with a spiral-spring-driven mechanical actuator. The device enables sequential execution of recombinase polymerase amplification (RPA), CRISPR-Cas12a-mediated detection, and lateral flow assay (LFA) readout through a single user-initiated winding step, without the need for external power or instrumentation. The multistep process was achieved through automatic reconfiguration of the paper pads, enabling pipette-free transfer of RPA amplicons from the paper-based RPA pad to the paper-based CRISPR pad, followed by transfer of the reporter probes to the LFA strip. The paper-based system is preloaded with lyophilized reagents and designed to autonomously control fluid flow and reaction timing. Targeting the vvhA gene specific to V. vulnificus, the platform achieved a limit of detection of 1 colony-forming unit per reaction (CFU/reaction) in pure cultures. This sensitivity was consistently reproduced in gill swab samples from oysters exposed to artificially contaminated seawater at 20 CFU/mL, confirming that reliable detection was achievable in oyster gill swabs under these conditions. Overall, this work establishes a fully mechanical, instrument-free RPA/CRISPR-based diagnostic system for V. vulnificus, offering a rapid, portable, and highly sensitive approach for point-of-care testing and food safety applications.