该研究构建包装饮用水中铜绿假单胞菌定性检测方法并开展应用效果评价。通过低菌浓度接种，选取合适培养条件及增菌基础肉汤，通过单因素试验优化增菌液配方，通过多重比较优化选择分离培养基配方。以GB 8538-2022方法为对照，选取市售包装饮用水加入不同类别、菌量标准菌后，使用臭氧发生器分别通气0、30、60、90 min后，再进行膜过滤富集增菌-选择划线分离-MALDI-TOF MS仪鉴定的定性测试，比较两者的灵敏度、选择性、特异性，并选取不同类别样品进行应用效果测试。该研究构建的定性检测方法第一步增菌选取TSB添加甘露醇4 g/L配方，42 ℃培养6~8 h；第二步选择分离选取CN添加丙酮酸钠0.20 g/L配方，42 ℃培养24~48 h，利用MALDI-TOF MS仪对可疑菌可实现准确、快速、高通量鉴定。该法对铜绿假单胞菌的最低识别限为3 CFU/250 mL，可识别1 000~10 000 CFU/250 mL干扰菌，对不同样品的检测识别效果较现行国标差异显著（x2=25.45，P<0.05），相对现有方法，具有灵敏度高、特异性强、简便安全、高效等技术优势。

A qualitative detection method for Pseudomonas aeruginosa in bottled water developed and its performance was evaluated. Appropriate cultivation conditions and meat-based enrichment broth were selected according to the inoculation results at low bacterial concentrations. Moreover, a single-factor test was performed to optimize the enrichment medium formula for inoculation at low bacterial concentrations. Thereafter, a multiple comparison test was conducted to optimize the solid medium for selection and separation. Commercially available bottled water was used and spiked with different concentrations of reference strains, followed by ozone addition for 0, 30, 60 and 90 min. The pre-treated water was then tested using the proposed qualitative detection method. More specifically, 4 g/L mannitol was added to tryptic soy broth (TSB). The bacteria were cultivated in liquid media at 42 ℃ for 6~8 h. Next, sodium 0.20 g/L pyruvate was added to CN agar and cultured at 42 ℃ for 24~48 h. Finally, MALDI-TOF MS was adopted to ensure accurate, rapid, and high-throughput detection of potentially pathogenic bacteria, including P. aeruginosa. The results were compared to those obtained via the GB 8538-2022 method in terms of sensitivity, selectivity, and specificity. Different types of samples were also selected to evaluate the wide range of applications that can be assessed using the proposed method. The results demonstrated that the proposed method can identify P. aeruginosa at concentrations as low as 3 CFU/250 mL. Moreover, it identified interfering bacteria at a concentration range of 1 000~10 000 CFU/250 mL. The detection and identification performances for different samples were statistically significant (x2=25.45, P<0.05). Thus, the method established in this study exhibits high sensitivity and specificity, ease of use, and high efficiency compared to existing methods.

广东省自然科学基金项目（2020A1515011561）