Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities
Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities
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DOI：
10.1038/ismej.2007.97
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发表时间：
2008-04-01
期刊：
影响因子：
11
通讯作者：
Joint, Ian
中科院分区：
文献类型：
--
作者：
Muhling, Martin;Woolven-Allen, John;Joint, Ian
门和类特异性PCR引物进行了测试的克隆库的生产和复杂的细菌群落的变性梯度凝胶电泳（DGGE）分析。设计引物以特异性扩增拟杆菌门、浮游菌门和厚壁菌门、变形菌门的三个类别（α-变形菌、β-变形菌和γ-变形菌）和蓝细菌的16 S rRNA基因片段（包括叶绿体16 S rRNA基因）。通过从中营养（挪威沿海）和贫营养（北方大西洋环流）环境的环境DNA样本中产生克隆库来测试这七对引物的特异性。7对引物中有5对特异性扩增16 S rRNA基因序列。β-变形菌和厚壁菌的特异性引物，这是相对成功的沿海水域围隔样品，但不那么与北方大西洋环流样品。从Gammaproteobacteria克隆库的序列的系统发育分析显示，沿海样品产生了一些属于寡营养海洋Gammaproteobacteria（OMG）组的分支内的集群的克隆，这表明该组并不局限于寡营养环境。采用两步或三步巢式PCR-DGGE方法比较了沿海和外海环境DNA样品的细菌多样性，发现细菌群落存在显着差异。组特异性引物的应用提供了比现有DGGE引物组更高分辨率的遗传指纹分析方法。
Phylum- and class-specific PCR primers were tested for the production of clone libraries and for denaturing gradient gel electrophoresis (DGGE) analysis of complex bacterial communities. Primers were designed to specifically amplify 16S rRNA gene fragments of the phyla Bacteroidetes, Planctomycetes and Firmicutes, of three classes of the phylum Proteobacteria, the Alphaproteo-bacteria, Betaproteobacteria and Gammaproteobacteria, and of the Cyanobacteria (including chloroplast 16S rRNA genes). The specificity of the seven primer pairs was tested by producing clone libraries from environmental DNA samples from mesotrophic (Norwegian coastal) and oligotrophic (Northern Atlantic Gyre) environments. Five of the seven primer pairs specifically amplified target 16S rRNA gene sequences. Exceptions were the Betaproteobacteria- and Firmicutes-specific primers, which were relatively successful with coastal water mesocosm samples but less so with the Northern Atlantic Gyre sample. Phylogenetic analysis of sequences from the Gammaproteobacteria clone library revealed that the coastal sample yielded a number of clones that clustered within clades that belong to the oligotrophic marine Gammaproteobacteria (OMG) group, indicating that this group is not confined exclusively to the oligotrophic environment. Comparison of the bacterial diversity of the environmental DNA sample from the coastal and the open ocean using a two- or three-step nested PCR-DGGE process revealed significant differences in the bacterial communities. The application of the group-specific primers provides a higher resolution genetic fingerprinting approach than existing DGGE primer sets.