Pseudomonas aeruginosa is a common opportunistic pathogen that causes acute nosocomial necrotizing pneumonia and is the predominant source of chronic lung infections in patients with the genetic disorder cystic fibrosis. Early diagnosis in infected patients and monitoring P. aeruginosa contamination is therefore of great importance in controlling disease spread and development with timely drugs intervention treatment and cut off infection source. Traditional culture-biochemical methods are time consuming and highly dependent on technicians and expensive instruments. To address these challenges, the present study aimed to develop a rapid, sensitive, and specific, on-site detection method for P. aeruginosa based on recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) technology. The experimental process included screening and modification of primer and probe sets targeting the unique virulence gene elastase B (lasB); specificity detection in 29 strains of P. aeruginosa and 23 closely-related pathogenic bacteria; sensitivity measurements with gradient-diluted P. aeruginosa genomic DNA and probit regression analysis; and clinical application evaluation using 574 patients samples and calculating coincidence rate and kappa index value in comparison with the culture-biochemical method. The P. aeruginosa RPA-LFS assay could complete the amplification process at 37°C constant temperature within 30 min and results could be visualized by the naked eye within 10 min on LFS. The assay displayed high sensitivity with a limit of detection of 3.05 CFU/reaction. It also demonstrated high specificity by showing no cross reaction with other pathogenic bacteria, and rapidness by being completed in less than an hour. Furthermore, when used with clinical samples, the assay had a coincidence rate of 98.26% with the culture-biochemical method and a kappa index value of 0.9433. These data indicate that the RPA-LFS assay represents a major improvement for P. aeruginosa detection, especially in resource-limited areas.

铜绿假单胞菌是一种常见的机会性病原体，可导致急性医院内坏死性肺炎，是遗传性疾病囊性纤维化患者慢性肺部感染的主要来源。感染患者的早期诊断和监测铜绿假单胞菌因此，及时进行药物干预治疗，切断感染源，污染对控制疾病传播和发展具有重要意义。传统的培养-生化方法耗时且高度依赖技术人员和昂贵的仪器。为了应对这些挑战，本研究旨在开发一种快速、灵敏、特异的现场检测方法铜绿假单胞菌基于重组酶聚合酶扩增（RPA）结合侧向流动条（LFS）技术。实验过程包括针对独特毒力基因的引物和探针组的筛选和修饰弹性蛋白酶B(激光); 29株的特异性检测铜绿假单胞菌23个密切相关的病原菌；梯度稀释的灵敏度测量铜绿假单胞菌基因组 DNA 和概率回归分析；临床应用评价采用574例患者样本，计算符合率和kappa指数值，与培养-生化法进行比较。这铜绿假单胞菌RPA-LFS检测可在37°C恒温下30分钟内完成扩增过程，在LFS上10分钟内肉眼即可看到结果。该测定显示出高灵敏度，检测限为 3.05 CFU/反应。它还表现出与其他病原菌没有交叉反应的高特异性，并且在不到一个小时内完成快速。此外，当用于临床样本时，该测定与培养-生化方法的符合率为98.26%，kappa指数值为0.9433。这些数据表明，RPA-LFS 测定代表了对铜绿假单胞菌检测，尤其是在资源有限的地区。