【作者】 曾海燕；

【导师】 王丽； 朱杰；

【作者基本信息】 华南农业大学 ， 工程硕士（专业学位）， 2023， 硕士

【摘要】 铜绿假单胞菌是广泛存在于水体的革兰氏阴性条件致病菌,有抗性强、营养要求低等特点,易引发婴幼儿、免疫缺陷或低下人群急性肠道炎及其他炎症。目前各地在流通环节中抽检包装饮用水发现铜绿假单胞菌的阳性率居高不下,且存在耐药性。随着检验量激增,现行GB 8538-2016受试剂质量和特性等因素影响,其灵敏度低、特异性差、检验周期长、可疑菌鉴定效果不佳等缺点日益凸显。因此,建立准确、简单、高通量筛查鉴定的检测方法对于包装饮用水中铜绿假单胞菌的污染防控有重要意义。本研究通过构建低菌量污染并长期储存的包装饮用水研究模型,建立在优化培养基营养和选择性的基础上,利用MALDI-TOF MS仪进行高通量鉴定的定性检测方法,并开展了应用效果评价。研究内容和结果如下:(1)包装饮用水模拟加菌放置变化测试。取三种类型水产品分别添加高、中、低菌浓度的铜绿假单胞菌及其与大肠埃希氏菌的混菌,30℃下放置35～80天,通过检测活菌数变化,发现纯净水菌数10天内下降较快,放置1月后趋近于0 CFU/250 m L;而矿物质水和天然水类型的水产品初始受到低浓度(0～10 CFU/250 m L)铜绿假单胞菌污染时,在80天内水样仍存在100～500 CFU/250 m L菌量的污染。因此,将包装饮用水的检测方法从定量的直接选择培养变为先增菌后分离的定性检验,可有效避免漏检。(2)优化增菌条件与培养基配方。确定了第一步液体增菌的培养条件为42℃培养6～8 h;经多重比较选取TSB添加甘露醇4 g/L作为增菌液,可使接种量为0～10CFU/m L低浓度的铜绿假单胞菌在较短时间内快速增长至10~2CFU/m L以上;经多重比较选取CN添加丙酮酸钠0.2 g/L作为第二步的选择分离用固体培养基,对目标菌的促生长效果、识别效果和对非目标菌的抑制效果均显著优于国标CN琼脂。(3)确定MALDI-TOF MS仪对不同状态铜绿假单胞菌有较好的鉴定效果。通过对模拟生产中经不同处理后受损的铜绿假单胞菌的鉴定,发现该方法能够准确、快速地对菌株进行鉴定。(4)开展膜过滤富集增菌-选择培养基划线分离-利用MALDI-TOF MS仪鉴定的定性方法应用效果评价。以GB8538-2016方法为对照,选取天然矿泉水加入不同类别、不同菌量的标准菌,验证该方法的灵敏度、选择性和特异性,并选取不同类型的样品进行实际应用效果测试。结果表明,该法对铜绿假单胞菌的最低识别限为3 CFU/250m L;同时可识别1000～10000 CFU/250 m L干扰菌;对不同样品的检测识别效果较现行国标差异显著(χ~2=25.45,p<0.05),利用MALDI-TOF MS仪对可疑菌可实现准确、快速、高通量鉴定,有效避免假阴性结果。本研究建立的检测方法相对现有方法,具有灵敏度高、特异性强、高效等技术优势。更多还原

【Abstract】 Pseudomonas aeruginosa is a gram-negative opportunistic pathogen widely present in aquatic environment and has the characteristics of strong resistance and low nutritional requirements.It is prone to cause acute enteritis and other inflammations in infants,immunodeficiency or people with low immunity.For now,the detection rate of Pseudomonas aeruginosa remains high in the sampling inspection of packaged drinking water in the circulation link,and the drug resistance is also present.With the surge in inspection volume,GB8538-2016,the existing detection method has defects such as low sensitivity,poor specificity,long inspection cycle and poor identification of target bacteria.Therefore,establishing assays for accurate,simple,high-throughput identification is of great significance for the prevention and control of Pseudomonas aeruginosa in packaged drinking water.In this study,by constructing a research model of packaged drinking water with low bacterial content and long-term storage,on the basis of optimizing the nutrition and selectivity of the medium,by using MALDI-TOF MS for high-throughput identification,a new methods for qualitative detection of Pseudomonas aeruginosa was established and the application effect evaluation was also evaluated.The main contents and results ara as follows.Constructing a research model of packaged drinking water with a certain amount of bacteria and long-term storage.Three types of packaged drinking water were added with high,medium and low amounts of Pseudomonas aeruginosa and its mixed with Escherichia coli,placed at 30°C for 35 to 80 days.According to the changes in the amount of bacteria detected,it was found that the number of bacteria in purified water decreased rapidly within 10 days,and approached 0 CFU/250 m L after being placed for 1 month.While in mineral water and natural water,with low initial bacterial content of Pseudomonas aeruginosa(0～10 CFU/250 m L),the cells with a increase concentration of 100 to 500CFU/250 m L within 80 days was detected.Therefore,changing the detection method in packaged drinking water from the quantitative method of direct culture to the qualitative method of first enrichment then separation can effectively avoid missed and false detection.Optimize the cultivate conditions and medium.The culture conditions of liquid enrichment in the first step were determined to be cultured at 42℃for 6h-8h.Optimized by complete randomalized design test,adding mannitol 4 g/L to TSB was selected as the enrichment medium.It has been proved that the number of bacteria increased rapidly from0-10 CFU/m L to more than 10~2CFU/m L in a relatively short period of time.Optimized by multiple comparisons test,adding sodium pyruvate 0.2 g/L to CN Agar was selected as the solid medium for the second step of selection and separation,which had better growth-promoting and identification effect than CN Agar for target bacteria,and also had better inhibitory effect for non-target bacteria.It was confirmed that MALDI-TOF MS has a good identification effect on Pseudomonas aeruginosa in different states.Through the identification of Pseudomonas aeruginosa in different treatments in the simulated process of production,it is found that this method give more accurately and rapidly efficiency on bacteria identification.In this study,membrane Filtration and enrichment and plating out combined identification by MALDI-TOF MS assays for qualitative detection of Pseudomonas aeruginosa in packaged drinking water were established and compared.Using the method in GB8538-2016 as the control,the natural mineral water was selected and added with different types and amounts of reference strains to verify the sensitivity,selectivity and specificity of the method.Different types of samples were also selected to test the actual application effect of the method.The results showed that the method can identify Pseudomonas aeruginosa as low as 3 CFU/250 m L.At the same time,it also identified interfering bacteria with a concentration of 1000～10000 CFU/250 m L.The detection and recognition effect of different samples was statistically significant(χ~2=25.45,p<0.05).It has accurate,rapid and high-throughput identification effect for suspicious bacteria by using MALDI-TOF MS,also avoiding false negative results effectively.It can be concluded that the qualitative detection method established in this study has some technical advantages of high sensitivity,strong specificity and high efficiency.更多还原