A Rapid and Sensitive CRISPR-Cas12a for the Detection of Legionella pneumophila.

Legionella pneumophila is a common environmental bacterium that can cause severe respiratory disease. In this study, a reliable, rapid, and convenient detection method for L. pneumophila was established using a combination of recombinase polymerase amplification (RPA) and CRISPR/Cas12a technology. First, we designed three pairs of RPA primers and two types of crRNA based on the L. pneumophila-specific mip gene. Subsequently, we optimized the primers and amplification time for the RPA reaction, the crRNA for the CRISPR/Cas12a reaction, as well as the concentration of the fluorescent probe. We successfully constructed an RPA-CRISPR/Cas12a fluorescence detection system and a portable RPA-CRISPR/Cas12a LFB. The detection systems achieved a sensitivity of 5 copies/μl and high specificity. One hundred sixty environmental water samples tested by RPA-CRISPR/Cas12a LFB showed no significant difference compared to the qPCR method, providing a reliable tool for future on-site detection.