Rapid, sensitive, and user-friendly detection of Pseudomonas aeruginosa using the RPA/CRISPR/Cas12a system

Abstract

Background: Pseudomonas aeruginosa (P. aeruginosa) is a life-threatening bacterium known for its rapid development of antibiotic resistance, posing significant challenges in clinical treatment, biosecurity, food safety, and environmental monitoring. Early and accurate identification of P. aeruginosa is crucial for effective intervention.

Methods: The lasB gene of P. aeruginosa was selected as the target for the detection. RPA primers for recombinase polymerase amplification (RPA) and crRNA for CRISPR/Cas12a detection were meticulously designed to target specific regions within the lasB gene. The specificity of the RPA/CRISPR/Cas12a detection platform was assessed using 15 strains. The detection limit of RPA/CRISPR/Cas12a detection platform was determined by utilizing a pseudo-dilution series of the P. aeruginosa DNA. The practical applicability of the RPA/CRISPR/Cas12a detection platform was validated by comparing it with qPCR on 150 samples (35 processed meat product samples, 55 cold seasoned vegetable dishes, 60 bottled water samples).

Results: The RPA/CRISPR/Cas12a detection platform demonstrates high specificity, with no cross-reactivity with non-P. aeruginosa strains. This assay exhibits remarkable sensitivity, with a limit of detection (LOD) of 10⁰ copies/µL for fluorescence assay and 10¹ copies/µL for the LFTS method. Furthermore, the performance of the RPA/CRISPR/Cas12a detection platform is comparable to that of the well-established qPCR method, while offering advantages such as shorter reaction time, simplified operation, and reduced equipment requirements.

Conclusions: The RPA/CRISPR/Cas12a detection platform presents a straightforward, accurate, and sensitive approach for early P. aeruginosa detection and holds great promise for diverse applications requiring rapid and reliable identification.

Keywords: Pseudomonas aeruginosa; CRISPR/Cas12a; Detection; Recombinase polymerase amplification.

Fig. 1

Schematic illustration of the principle of the lateral flow test strip. (A) Following RPA amplification, the samples undergo CRISPR/Cas12a cleavage reaction. (B) The test strip displays the positions of gold nanoparticles, anti-FITC antibody, streptavidin (SA), and goat anti-mouse IgG. (C) The complexes of gold nanoparticle-anti-FITC antibody-ssDNA reporter are captured by SA, resulting in the visibility of the C line. (D) In the presence of the target, the complexes of FITC-anti-FITC antibody-gold nanoparticle are captured by the goat anti-mouse IgG, thereby making the T line visible

Fig. 2

The design sites of the RPA primers and crRNA

Fig. 3

The fluorescence curves generated by CRISPR/Cas12a detection reaction. crRNA 1: the reaction system with crRNA1 addition, crRNA 2: the reaction system with crRNA2 addition

Fig. 4

Specificity assay of the RPA/CRISPR/Cas12a detection platform. (A) Background - subtracted fluorescence intensity of fluorescence assay method, values represent the mean ± SD (n = 3). (B) The outcomes of the LFTS method. 1. P. aeruginosa standard strain, 2–5. P. aeruginosa clinically isolated strains, 6. P. stutzeri, 7. P. putida, 8. P. alcaligenes, 9. P. fluorescens, 10. Staphylococcus aureus, 11. Escherichia coli, 12. Shigella flexner, 13. Listeria monocytogenes,14. Salmonella typhimurium, 15. Bacillus subtilis

Fig. 5

Sensitivity of the RPA/CRISPR/Cas12a detection platform (A) The histogram of background-subtracted fluorescence intensity obtained from different DNA concentration of P. aeruginosa, values represent the mean ± SD (n = 3). (B) The result of the LFTS method