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

Area of Science:

• Biotechnology
• Microbiology
• Medical Diagnostics

Background:

• Vibrio vulnificus is a dangerous marine pathogen causing severe infections with high mortality.
• Current detection methods require laboratory equipment and trained staff, hindering rapid, on-site diagnostics.
• There is a critical need for portable, sensitive, and user-friendly detection systems for V. vulnificus.

Purpose of the Study:

• To develop an automated, paper-based diagnostic platform for V. vulnificus detection.
• To integrate recombinase polymerase amplification (RPA) and CRISPR-Cas12a systems into a single, instrument-free device.
• To enable rapid, sensitive, and on-site detection of V. vulnificus without external power or instrumentation.

Main Methods:

• A microfluidic paper pad device with a spiral-spring actuator was designed for automated reagent handling and fluid flow.
• The platform sequentially performs RPA, CRISPR-Cas12a detection, and lateral flow assay (LFA) readout.
• Lyophilized reagents were preloaded, and the system autonomously controlled fluid transfer and reaction timing for pipette-free operation.

Main Results:

• The RPA/CRISPR-Cas12a platform achieved a limit of detection of 1 colony-forming unit per reaction (CFU/reaction) for V. vulnificus.
• Sensitive detection was validated in artificially contaminated oyster gill swab samples at 20 CFU/mL.
• The fully mechanical, instrument-free system demonstrated reliable performance in complex sample matrices.

Conclusions:

• A novel, fully mechanical, instrument-free diagnostic system for V. vulnificus was successfully developed.
• The paper-based RPA/CRISPR-Cas12a platform offers a rapid, portable, and highly sensitive detection solution.
• This technology holds significant promise for point-of-care testing and enhancing food safety surveillance.