铜绿假单胞菌重组酶聚合酶扩增检测方法研究

Study on a Recombinase Polymerase Amplification Assay for Rapid Detection of Pseudomonas aeruginosa

基于铜绿假单胞菌中高度保守的外毒素A基因设计特异性引物,建立并优化重组酶聚合酶扩增(Recombinase Polymerase Amplification,RPA)条件,并评价该方法的灵敏度和抗干扰能力。结果显示,RPA反应体系能够在40℃等温条件下,20 min内实现对目标基因的扩增,检出铜绿假单胞菌的灵敏度为10~2 CFU·mL^(-1),抗干扰能力与GB 8538-2022相一致。经培养优化后,铜绿假单胞菌的最低检出限可达10 CFU·mL^(-1),且重复性良好。本研究探索的铜绿假单胞菌RPA扩增检测方法操作简单、反应迅速、抗干扰能力强,为食品中铜绿假单胞菌的快速识别提供了新的方向与参考。

Based on the highly conserved exotoxin A gene in Pseudomonas aeruginosa,specific primers were designed to establish and optimize the amplification conditions of recombinase polymerase amplification (RPA),the sensitivity and anti-interference ability of the method were evaluated.The results showed that the RPA reaction system could achieve the amplification of the target gene within 20 minutes under the isothermal condition of 40 ℃,and the sensitivity of detecting Pseudomonas aeruginosa was 10~2 CFU·mL^(-1),and the anti-interference ability was consistent with GB 8538-2022.After culture optimization,the minimum detection limit of Pseudomonas aeruginosa could reach 10 CFU·mL^(-1),and the repeatability was good.The RPA amplification detection method of Pseudomonas aeruginosa established in this study has the advantages of simple operation,rapid response and strong anti-interference ability,which provides a new direction and reference for the rapid detection of Pseudomonas aeruginosa in food.