【摘要】 为了直接检测环境水体中军团菌的存在,文章将巢式PCR与倍比稀释法相结合(巢式PCR-MPN法),采用两对引物扩增环境样品基因组DNA。将检测到含有军团菌的样品DNA模板进行十倍梯度稀释,由最低检测的稀释度,计算出样品中军团菌的细胞数,同时确定方法检测的灵敏度。并用克隆建库、构建系统树的方法来验证该文研究方法的特异性。研究结果表明,巢式PCR第二步后获得的序列均为军团菌序列,该方法军团菌DNA的检出限可以低至2.74 fg/μL。该研究建立的水体军团菌检测方法具有简便快捷、特异性好、灵敏度高的特点。更多还原

【Abstract】 To directly identify the presence of Legionella in freshwater environment,a nest-PCR-MPN method was set up,which combining nested polymerase chain reaction(nest-PCR)with most probable number(MPN)method.The nest-PCR assay applied two pairs of primers to amplify 16S rRNA gene of Legionella spp.in freshwater.In the positive amplified samples,detection limit of the method was evaluated by using decimal dilutions of genomic DNA until the nest-PCR was negative.Clone libraries and phylogenetic affiliation analysis were also used for verifying the specificity of the method.Results showed that the established method was specific to detect Legionella spp.from environmental samples,and the detection limit could reach 2.74 fg/μL of Legionella DNA.更多还原