Single-tube two-step RPA-CRISPR/Cas12b platform for detection of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen of significant clinical and public health concern, necessitating the development of rapid and reliable detection methods. Traditional diagnostic approaches, which rely on culture-dependent techniques and biochemical identification, are often labor-intensive, time-consuming, and technically demanding. This study describes a novel single-tube, two-step, rapid detection platform that integrates recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats-associated protein Cas12b technology. Through systematic experimental optimization, the study identified an optimal RPA primer pair (F2-R1) and single-guide ribonucleic acid 553 that targets the lasR gene of P. aeruginosa, with reaction conditions optimized at 42°C and a primer concentration of 10 μM. The RPA-clustered regularly interspaced short palindromic repeats/Cas12b fluorescence detection system (RPA-Cas12b-Fluo) demonstrated a sensitivity threshold of 10 copies of deoxyribonucleic acid (DNA) per reaction and a bacterial detection limit of 50 colony-forming units (CFU) per reaction. When coupled with a lateral flow strip (RPA-Cas12b-LFS), the sensitivity was slightly reduced but remained robust, achieving detection limits of 10² copies and 200 CFU per reaction. Specificity assays confirmed a high discriminatory capacity for P. aeruginosa with no cross-reactivity observed against P. fluorescens, P. putida, or six common foodborne pathogens, thereby validating the specificity profile of the platform. The applicability of the method was further validated by analyzing 20 water samples, which demonstrated 100% concordance with the national standard culture method. These findings have significant implications for improving outbreak surveillance and mitigating the risk of foodborne transmission associated with P. aeruginosa.