环境水体中空肠弯曲杆菌荧光定量PCR快速检测方法的研究

发布时间：2018-08-04 21:25

【摘要】：目的建立一种环境水体中空肠弯曲杆菌的荧光定量PCR准确快速方法。方法采用膜吸附-洗脱法浓缩富集环境水体中的空肠弯曲杆菌,根据空肠弯曲杆菌VS1基因序列设计引物,以构建的空肠弯曲杆菌重组质粒作为标准品,建立空肠弯曲杆菌荧光定量PCR检测方法。结果荧光定量PCR反应体系在2.66×109～2.66×100copies/μl拷贝数模板量的线性范围内,所获得回归方程线性关系较好(R2=0.998),扩增曲线呈S形指数增长;特异性检测中肠炎沙门氏菌等对照菌扩增呈阴性,对于空肠弯曲杆菌纯培养物检测限达到46 cfu/ml,实际水样检测结果与常规生化鉴定结果一致,整个检测过程可以在4 h内完成。结论本研究中建立的空肠弯曲杆菌荧光定量PCR检测方法具有准确、简便、快速等特点,具有较强的实际应用价值,可以用于环境水体中空肠弯曲杆菌的检测。
[Abstract]:Objective to establish an accurate and rapid method for fluorescence quantitative PCR of Campylobacter jejuni in environmental water. Methods the membrane adsorption-elution method was used to concentrate and enrich Campylobacter jejuni in environmental water. Primers were designed according to the VS1 gene sequence of Campylobacter jejuni. The recombinant plasmid of Campylobacter jejuni was used as the standard. A fluorescence quantitative PCR method for detection of Campylobacter jejuni was established. Results in the linear range of 2.66 脳 10 ~ 9 ~ 2.66 脳 100copies/ 渭 l copy number template, the regression equation was linear (R _ 2N _ (0.998), and the amplification curve was increased by S-shaped index, and the amplification of Salmonella enteritidis and other control bacteria was negative in the specific detection. For the pure culture of Campylobacter jejuni, the detection limit was up to 46cf / ml. The result of water sample detection was consistent with that of routine biochemical identification, and the whole detection process could be completed within 4 hours. Conclusion the fluorescent quantitative PCR method for the detection of Campylobacter jejuni in this study is accurate, simple and rapid, and has strong practical application value. It can be used for the detection of Campylobacter jejuni in environmental water.
【作者单位】：解放军后勤工程学院军事油料应用与管理工程系;国防建筑规划与环境工程系;
【基金】：国家质量监督检验检疫总局科研项目(200910140-03) 中国人民解放军总后勤部基建营房部项目(BY111C012)
【分类号】：R378

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