Detection of waterborne Pseudomonas aeruginosa monovalent serogroup E using the ultrasensitive HDA-CRISPR/Cas12a method

The nucleic acid isothermal amplification method allows for the highly sensitive detection of the important pathogen Pseudomonas aeruginosa ( P. aeruginosa ), providing crucial information for controlling pathogen transmission. In this study, we exploited the high GC content of a specific target within the monovalent serogroup E of P. aeruginosa , and through the design of primers and CRISPR RNA (crRNA) sequences, achieved robust exponential amplification of the target DNA. We integrated the HDA detection system with an optimized CRISPR/Cas12a, resulting in the development of the novel "HDA-Cas12aFQ" platform, which enables detection within 2 hours. Validation experiments confirmed that the HDA-Cas12aFQ platform exhibits high specificity and sensitivity for detecting P. aeruginosa monovalent serogroup E in serum samples. Specifically, the platform demonstrated a detection limit of 10 2 CFU/mL for pure cultures, a DNA sensitivity of 0.695 pmol/L—nearly 200 times greater than that of the real-time HDA method. Additionally, the positive detection rate of real-world samples was in strong agreement with that of traditional, well-established detection methods. The HDA-Cas12aFQ platform is expected to become a leading method for ensuring drinking water purity due to its exceptional sensitivity and accuracy, thus playing a crucial role in safeguarding public health. • Development of a novel HDA-Cas12aFQ platform for rapid pathogen detection. • High sensitivity and specificity achieved for P. aeruginosa serogroup E. • The HDA-Cas12aFQ platform sensitivity was augmented to 0.695 pmoL/L, a 200-fold increase. • Streamlined detection process completed in just 2 hours. • Enhanced efficiency in monitoring drinking water safety and quality.