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Development of a Highly Specific RPA/CRISPR-Cas13a Assay for Detection of Pseudomonas aeruginosa Virulence Factor
Lucía Ceballos-Romero1,2, Soraya Herrera-Espejo3, Daniel Atassi3
1Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, 28029 Madrid, Spain.
Abstract:
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.
Insights
A new diagnostic test accurately detects Pseudomonas aeruginosa and its ExoU virulence factor in blood. This rapid method, combining recombinase polymerase amplification and CRISPR-Cas13a, shows promise for faster clinical decisions in treating infections.
Area of Science:
- Molecular diagnostics
- Microbiology
- Infectious diseases
Background:
- Rapid and accurate detection of Pseudomonas aeruginosa is critical for effective patient management.
- The ExoU virulence factor contributes significantly to P. aeruginosa pathogenicity.
- Existing diagnostic methods may lack the speed and specificity required for timely intervention.
Purpose of the Study:
- To develop a novel diagnostic assay for the simultaneous detection of P. aeruginosa and its exoU gene.
- To evaluate the sensitivity, specificity, and performance of the assay in clinical samples like blood.
Main Methods:
- Development of a CRISPR-Cas13a-based diagnostic assay.
- Integration of recombinase polymerase amplification (RPA) for nucleic acid amplification.
- Utilisation of a fluorescence-based readout for signal detection and discrimination.
Main Results:
- The RPA/CRISPR-Cas13a assay achieved robust amplification and detection.
- Sensitivity reached 6 log10 CFU/mL in Luria-Bertani medium and 8 log10 CFU/mL in blood.
- The assay demonstrated 100% specificity, with no cross-reactivity against other bacterial strains.
Conclusions:
- The developed RPA/CRISPR-Cas13a assay shows significant potential for rapid P. aeruginosa and ExoU identification.
- This diagnostic approach could expedite clinical decision-making for infections caused by P. aeruginosa.
- Further clinical validation is necessary to establish its role as a bedside diagnostic tool.
