A Rapid RPA-CRISPR/Cas12a Approach for Detecting Mycobacterium marinum.

Mycobacterium marinum is one of the most common atypical mycobacteria causing opportunistic infections in humans. It is considered to be one of the most important fish pathogens and is associated with a variety of diseases in freshwater and marine fish. In this study, a rapid detection method for M. marinum was established based on the Recombinase Polymerase Amplification (RPA) technology and the CRISPR/Cas12a trans-cut principle. By designing and screening RPA primers and crRNA, the RPA amplification system and CRISPR/Cas12a detection system were optimized, and an RPA-CRISPR/Cas12a detection system with high sensitivity and specificity was successfully constructed. The LOD95 of this detection method was estimated to be 5.7 copies/μL (95% CI: 2.4-15.5 copies/μL). The RPA-CRISPR assay showed perfect agreement with qPCR, with 100% sensitivity (95% CI: 76.8%-100%) and 100% specificity (95% CI: 73.5%-100%). The RPA-CRISPR/Cas12a-M. marinum detection method established in this study has the advantages of simple operation and visualization of results. It provides an effective tool for rapid on-site detection of M. marinum and is expected to play an essential role in the monitoring and prevention of aquaculture diseases.