Sequences of primers and probes.

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.铜绿假单胞菌（Pseudomonas aeruginosa）是一种广泛存在的条件致病菌，具有重大的临床与公共卫生关注度，因此亟需开发快速可靠的检测方法。传统诊断方法依赖于依赖培养的技术与生化鉴定手段，往往操作繁琐、耗时较长且对技术要求较高。本研究报道了一种新型单管双步法快速检测平台，该平台整合了重组酶聚合酶扩增（recombinase polymerase amplification，RPA）与成簇规律间隔短回文重复序列相关蛋白Cas12b（clustered regularly interspaced short palindromic repeats-associated protein Cas12b）技术。通过系统性实验优化，本研究筛选出靶向铜绿假单胞菌lasR基因的最优RPA引物对（F2-R1）与单向导核糖核酸（single-guide ribonucleic acid）553，并将反应体系优化为42℃、引物浓度10 μM。本研究构建的RPA-成簇规律间隔短回文重复序列/Cas12b荧光检测系统（RPA-Cas12b-Fluo），灵敏度阈值可达每反应体系10拷贝脱氧核糖核酸（deoxyribonucleic acid，DNA），细菌检测限为每反应体系50菌落形成单位（colony-forming units，CFU）。当该体系与侧向流试纸条（lateral flow strip）联用（RPA-Cas12b-LFS）时，灵敏度略有下降但仍保持优异性能，检测限可达每反应体系10²拷贝DNA与200 CFU。特异性实验结果显示，该平台对铜绿假单胞菌具有极高的区分能力，与荧光假单胞菌、恶臭假单胞菌及6种常见食源性致病菌均无交叉反应，验证了其特异性表现。通过对20份水样的检测，进一步验证了该方法的适用性，其检测结果与国家标准培养法完全吻合（吻合度达100%）。本研究结果对于加强铜绿假单胞菌暴发监测、降低其食源性传播风险具有重要意义。