Medical college, Henan University of Chinese Medicine, No.156, Jinshui East Road, Zhengzhou, 450046, Henan, China.
Autobio Diagnostics Co., Ltd., No.199, 15th Ave, Zhengzhou, 450016, Henan, China.
BMC Infect Dis. 2024 Apr 30;24(1):458. doi: 10.1186/s12879-024-09348-3.
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.
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).
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 10 copies/µL for fluorescence assay and 10 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.
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.
铜绿假单胞菌（P. aeruginosa）是一种对生命构成威胁的细菌，其对抗生素的耐药性发展迅速，给临床治疗、生物安全、食品安全和环境监测带来了重大挑战。早期、准确地识别 P. aeruginosa 对于进行有效的干预至关重要。
选择铜绿假单胞菌的 lasB 基因为检测目标。精心设计了用于重组酶聚合酶扩增（RPA）的引物和用于 CRISPR/Cas12a 检测的 crRNA，以针对 lasB 基因内的特定区域。利用 15 株菌评估了 RPA/CRISPR/Cas12a 检测平台的特异性。通过利用 P. aeruginosa DNA 的伪稀释系列来确定 RPA/CRISPR/Cas12a 检测平台的检测限。通过将 RPA/CRISPR/Cas12a 检测平台与 150 个样本（35 个加工肉产品样本、55 个冷调味蔬菜菜肴、60 个瓶装水样本）上的 qPCR 进行比较，验证了 RPA/CRISPR/Cas12a 检测平台的实际适用性。
RPA/CRISPR/Cas12a 检测平台具有高度特异性，与非铜绿假单胞菌菌株无交叉反应。该测定法具有显著的灵敏度，荧光测定法的检测限（LOD）为 10 拷贝/μL，LFTS 方法的 LOD 为 10 拷贝/μL。此外，RPA/CRISPR/Cas12a 检测平台的性能与成熟的 qPCR 方法相当，但具有反应时间更短、操作更简单、设备要求更低等优势。
RPA/CRISPR/Cas12a 检测平台提供了一种简单、准确、灵敏的早期铜绿假单胞菌检测方法，在需要快速可靠鉴定的各种应用中具有广阔的应用前景。
