Abstract

Vibrio alginolyticus is a halophilic marine bacterium that threatens aquaculture and food safety. Rapid and reliable detection is vital for early intervention and outbreak prevention. Recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12a offers a sensitive, specific, and isothermal alternative suitable for portable detection. We developed a one-tube RPA/Cas12a assay for rapid and sensitive V. alginolyticus detection, which integrates fluorescence and lateral flow strip (LFS) readouts for flexible visualization. An optimal primer pair (F1/R1) and crRNA2 targeting the toxR gene enabled efficient amplification and Cas12a-mediated trans-cleavage. All key components—RPA mixture, Cas12a, and crRNA—were essential for signal generation. Reaction optimization achieved a detection limit of 0.747 copies/µL for fluorescence and 100 copies/µL for LFS. Specificity assays confirmed the exclusive detection of V. alginolyticus without cross-reactivity to other Vibrio spp. or marine bacteria. The method validation was carried out using multiple types of samples, including artificially contaminated clinical samples, aquatic products, as well as clinical isolated of V. alginolyticus , demonstrating robustness. This dual-mode system simplifies operation, minimizes contamination, and enables field-deployable, real-time monitoring of V. alginolyticus in aquaculture environments, offering a practical, rapid, and instrument-free tool for pathogen surveillance and coastal biosecurity.