The development of RPA-CRISPR/Cas12a assay for the detection of Pseudomonas plecoglossicida

Pseudomonas plecoglossicida is the etiological agent of visceral white spot disease, which induces significant mortality in economically important fish such as the large yellow croaker. In this study, we integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection to establish a fluorescence-based assay for rapid identification of P. plecoglossicida. The complete single-tube, two-stage RPA-CRISPR/Cas12a workflow can be performed within ~45 min. Using purified genomic DNA, the assay achieved an analytical detection limit of 1.65 copies μl-1 and showed no cross-reactivity with several other common fish pathogens. Its applicability was further evaluated using crude DNA extracts from spleen, liver, and kidney tissues of experimentally infected large yellow croakers. Overall, with its rapid turnaround, minimal equipment requirement, and high sensitivity, the RPA-CRISPR/Cas12a assay represents a promising diagnostic tool for rapid detection of P. plecoglossicida, thereby helping to control the spread of infection.