Development of a Highly Specific RPA/CRISPR-Cas13a Assay for Detection of Pseudomonas aeruginosa Virulence Factor ExoU in Blood Samples

Rapid detection of Pseudomonas aeruginosa and its virulence factor ExoU is essential for improving patient outcomes. In this study, a CRISPR–Cas13a-based diagnostic assay combined with recombinase polymerase amplification (RPA) was developed to detect P. aeruginosa and the exoU gene in blood samples. The assay demonstrated robust amplification, with detection limits of 6 log10 and 8 log10 CFU/mL in Luria–Bertani medium and blood, respectively, and a 100% specificity, without cross-reactivity against four Gram-negative bacilli and Staphylococcus aureus reference strains. The utilisation of a fluorescence-based readout facilitated unambiguous discrimination between P. aeruginosa and P. aeruginosa/exoU+ isolates vs. negative controls. In conclusion, these results support the potential of RPA/CRISPR-Cas13a diagnostics for the rapid identification of P. aeruginosa and its ExoU virulence factor. Further optimisation and clinical validation are required to confirm its utility as a bedside diagnostic test, where its application would speed up clinical decisions in the treatment of these infections.