Improvement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxa
Improvement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxa
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DOI：
10.1016/j.mimet.2011.06.010
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发表时间：
2011-09-01
影响因子：
2.2
通讯作者：
Burgess, J. Grant
中科院分区：
文献类型：
--
作者：
De Gregoris, Tristano Bacchetti;Aldred, Nick;Burgess, J. Grant
绘制自然环境中系统发育上不同细菌的分布图对于理解生态动力学具有重要意义。在这里，我们提出了一种定量PCR （qPCR）方法，用于分析自然群落中较高的分类群组成，该方法通过允许在同一qPCR运行期间对多个分类群进行定量分析，从而提高了以前可用的方法。现有的针对厚壁菌门（Firmicutes）、放线菌门（Actinobacteria）、拟杆菌门（Bacteroidetes）和变形菌门(Proteobacteria) α亚群和γ亚群的16S rRNA基因的引物得到了改进，在不降低其特异性的情况下，大大增加了它们所针对分类单元的覆盖范围。qPCR方法在体外测试16S rRNA序列的人工混合物中得到了验证，并用于表征年轻海洋生物膜中自然群落的组成。讨论了所提出的技术在揭示影响高等细菌分类群的生态动力学方面的可能贡献。(C) 2011 Elsevier B.V.版权所有
Mapping the distribution of phylogenetically distinct bacteria in natural environments is of primary importance to an understanding of ecological dynamics. Here we present a quantitative PCR (qPCR) assay for the analysis of higher taxa composition in natural communities that advances previously available methods by allowing quantification of several taxa during the same qPCR run. Existing primers targeting the 16S rRNA gene specific for Firmicutes, Actinobacteria, Bacteroidetes and for the alpha and gamma subdivisions of the Proteobacteria were improved by largely increasing the coverage of the taxon they target without diminishing their specificity. The qPCR assay was validated in vitro testing artificial mixtures of 16S rRNA sequences and used to characterise the composition of natural communities developing in young marine biofilms. The possible contribution of the proposed technique in revealing ecological dynamics affecting higher bacterial taxa is discussed. (C) 2011 Elsevier B.V. All rights reserved.