包装饮用水中铜绿假单胞菌实时荧光定量PCR快速检测  

Study on the rapid detection of Pseudomonas aeruginosa by real-time fluorescence quantitative PCR in packaged drinking water

作　　者：王芳妹 徐越[1] 林丹 朱秋燕 洪振柏 蒋伟 WANG Fang-mei;XU Yue;LIN Dan;ZHU Qiu-yan;HONG Zhen-bai;JIANG Wei(Hunan Province Produced Commodity Quality Inspection Institute,Changsha,Hunan 410007,China;Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China,College of Life Science,Hunan Normal University,Changsha,Hunan 410081,China)

机构地区：[1]湖南省产商品质量检验研究院,湖南长沙410007 [2]湖南师范大学生命科学学院蛋白质化学与发育生物学教育部重点实验室,湖南长沙410081

出　　处：《食品与机械》2023年第6期75-80,共6页Food and Machinery

基　　金：湖南省市场监督管理局科技计划项目(编号:2022KJJH49)。

摘　　要：目的:有效弥补传统微生物学检验方法的缺陷。方法:针对GB 8538—2022中铜绿假单胞菌的毒力基因pcrL和16S rRNA基因的V3~V4区保守序列设计合成引物和探针,建立实时荧光PCR方法。结果:该方法可以有效扩增常见标准储备菌株的16S rRNA基因,且对铜绿假单胞菌标准储备菌株的毒力基因pcrL的检测效果良好,观察到明显的“S”型扩增曲线,但对常见的食源性致病菌无扩增曲线。当菌液浓度<10 CFU/mL时,pcrL基因仍有扩增曲线,其检测范围为10~10~7 CFU/mL,检测限为10 CFU/mL;不同菌液浓度下毒力基因pcrL的Ct平均值为18.0~38.6。对近3年湖南省14个县、市抽查检验(瓶)桶装水中铜绿假单胞菌不合格样品进行进一步检测,符合率达到100%。结论:该方法对铜绿假单胞菌携带毒力因子pcrL检测的灵敏度较高;且操作简便、特异性强,具有良好的实用性。Objective:The molecular biological test has the advantage of fast and sensitive,which can effectively make up for the defects of the traditional microbial test method.Methods:According to the conserved sequences of the V3~V4 region of the virulence gene pcrL and the universal gene 16S rRNA of Pseudomonas aeruginosa in GB 8538—2022,primers and probes were designed and synthesized,and a real-time fluorescent PCR kit was established in this study.Results:The results showed that the common standard reserve strain 16S rRNA was amplified,and the virulence gene pcrL was only effective for nucleic acid detection of Pseudomonas aeruginosa standard reserve strain,and obvious"S"type amplification curve could be observed,while no amplification curve was found for common food-borne pathogens in this study.Gene pcrL still has amplification curve,when the concentration of bacterial solution was less than 10 CFU/mL,the detection range was 10~107 CFU/mL,and the detection limit was 10 CFU/mL.The Ct mean value of virulence gene pcrL under different bacterial solution concentrations ranged from 18.0 to 38.6.The results showed that this method was highly sensitive to the detection of the virulence factor pcrL carried by Pseudomonas aeruginosa.In the past three years,the unqualified Pseudomonas aeruginosa samples in 14 counties and cities of Hunan Province were further detected,and the coincidence rate reached 100%.Conclusion:This method is highly sensitive to detect the virulence factor pcrL carried by Pseudomonas aeruginosa.It has the advantages of simple operation,strong specificity,high sensitivity and good practicability.

关 键 词：铜绿假单胞菌 实时荧光定量PCR 毒力基因 pcrL 16S rRNA 饮用水 

分 类 号：R123.1[医药卫生—环境卫生学]