饮用水大肠菌群Taqman-MGB荧光定量PCR检测方法研究  

STUDY ON THE TAQMAN-MGB FLUORESCENCE QUANTITATIVE PCR METHOD IN DETECTION OF COLIFORMS IN DRINKING WATER

作　　者：刘渠[1] 李涌[1] 陈应坚[1] 苏雪媚[2] 

机构地区：[1]深圳市龙岗区疾病预防控制中心,深圳518110 [2]深圳市龙岗区人民医院

出　　处：《现代预防医学》2010年第12期2312-2314,2317,共4页Modern Preventive Medicine

摘　　要：[目的]建立快速、灵敏、特异的饮用水大肠菌群荧光定量PCR定量检测方法。[方法]以大肠菌群的lacZ基因为检测靶基因,设计荧光定量PCR引物和Taqman-MGB探针,建立荧光定量PCR定量反应体系,并评价荧光定量PCR方法的灵敏度、特异性、重复性。[结果]建立大肠菌群的荧光定量PCR检测方法。25μl荧光定量PCR反应体系主要成分浓度分别为Mg2+5.0mmol/L,dNTPs0.2mmol/L,引物0.2μmol/L,探针0.5μmol/L,4×ROX参比荧光染料0.5μl,Taq DNA聚合酶2.0U,模板5μl。方法可检出每微升单个拷贝大肠菌群模板,方法的特异性、重复性良好。[结论]荧光定量PCR方法可快速、灵敏、特异地检出水质中大肠菌群,可为饮用水大肠菌群快速检测标准方法的建立提供参考。[Objective] To establish fluorescence quantitative PCR assay,which was specific and sensitive for rapidly detection of coliforms in drinking water.[Methods] lacZ gene of coli group was selected as target gene,the primers of HBVDNA and Taqman-MGB probe were designed,and the HBV-DNA of PCR system quantitative reaction system was established,and the sensitivity,specificity and reproducibility of method was evaluated.[Results] The fluorescence quantitative PCR method was well established.The best 25μl reaction components was 5.0mmol /L Mg^2 ＋,0.2mmol /L dNTPs,0.2 μmol /L primers,0.5 μmol /L probe,4×ROX Reference Dye 0.5μl,2.0U TaqDNase and 5μl template.The detection limit for coliforms was single copies in each microlitre of coliform template.The method showed good sensitivity,specificity and repeatability.[Conclusion] The fluorescence quantitative PCR method could rapidly and exactly detect the coliforms in drinking water,which could conducive to establish the standard method of coliform detection.

关 键 词：大肠菌群 荧光定量PCR LACZ 

分 类 号：R378.21[医药卫生—病原生物学]