湖泊中军团菌的巢式PCR-MPN检测法  

Development of a Nest-PCR-MPN Method for Detection of Legionella spp.in Freshwater Environment

作　　者：王娜[1,2] 刘庆[1] 邢鹏[2] 余多慰[1] 吴庆龙[2] 

机构地区：[1]南京师范大学生命科学学院,江苏南京210046 [2]中国科学院南京地理与湖泊研究所湖泊与环境国家重点实验室,江苏南京210008

出　　处：《环境科学与技术》2011年第2期107-110,共4页Environmental Science & Technology

基　　金：国家重点基础研究发展规划(973)项目(2008CB418104);中国科学院方向性项目(KZCX1-YW-14-1;KZCX2-YW-JC302);江苏省人才攀登计划项目(BK2009024)

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。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.

关 键 词：军团菌 巢式PCR-MPN法 湖泊 

分 类 号：X830.2[环境科学与工程—环境工程]