An advanced molecular strategy to identify bacterial communities on art objects
An advanced molecular strategy to identify bacterial communities on art objects
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DOI：
10.1016/s0167-7012(01)00227-5
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发表时间：
2001-06-01
影响因子：
2.2
通讯作者：
Rölleke, S
中科院分区：
文献类型：
--
作者：
Schabereiter-Gurtner, C;Piñar, G;Rölleke, S
基于分子生物学方法，特别是基于16S rRNA基因的PCR扩增的非培养依赖性技术的应用试图克服常规培养方法的相同缺点，并揭示了艺术品上比培养方法所能显示的复杂得多的细菌群落。通过分子手段研究艺术品上的微生物生长的主要挑战之一是DNA的提取，这是由于样品量小和PCR抑制剂。在本研究中，我们介绍了一种DNA提取方案，它允许提取的PCR扩增的DNA来自石灰壁画和壤土地下的样品。DNA提取物用于扩增16S核糖体片段，随后通过变性梯度凝胶电泳（DGGE）进行分析。与DGGE分析平行，构建含有核糖体基因的PCR片段的克隆文库，并通过DGGE筛选克隆。克隆文库允许在微生物的系统发育分析中包含整个16S rDNA序列，提供比从切除和直接测序的DGGE条带的序列分析获得的微生物的系统发育鉴定更可靠的微生物。(C)2001 Elsevier Science B.V.保留所有权利。
The application of culture-independent techniques based on molecular biological methods, especially on the PCR amplification of 16S rRNA genes, attempts to overcome same shortcomings of conventional cultivation methods and reveals far more complex bacterial communities on art objects than can be shown by cultivation methods. One of the major challenges of investigating microbial growth on art objects by molecular means is the extraction of DNA, due to small sample amounts and PCR inhibitors. In the present study, we introduce a DNA extraction protocol, which allowed the extraction of PCR-amplifiable DNA from samples derived from lime wall paintings and loamy soil underground. The DNA extracts were used to amplify 16S ribosomal fragments, which were subsequently analyzed by denaturing gradient gel electrophoresis (DGGE). In parallel with the DGGE analysis, clone libraries containing PCR fragments of the ribosomal gene were constructed and clones were screened by DGGE. Clone libraries allow the inclusion of the entire 16S rDNA sequence in the phylogenetic analyses of microorganisms, providing a more reliable phylogenetic identification of microorganisms than is obtained from sequence analyses of excised and directly sequenced DGGE bands. (C) 2001 Elsevier Science B.V. All rights reserved.