Nested PCR-denaturing gradient gel electrophoresis approach to determine the diversity of sulfate-reducing bacteria in complex microbial communities
Nested PCR-denaturing gradient gel electrophoresis approach to determine the diversity of sulfate-reducing bacteria in complex microbial communities
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DOI：
10.1128/aem.71.5.2325-2330.2005
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发表时间：
2005-05-01
影响因子：
4.4
通讯作者：
Muyzer, G
中科院分区：
文献类型：
--
作者：
Dar, SA;Kuenen, JG;Muyzer, G
在这里，我们描述了一种三步套式-聚合酶链式反应-变性梯度凝胶电泳法(DGGE)来检测工业生物反应器中复杂微生物群落中的硫酸盐还原细菌(SRB)。在第一步中，用细菌引物扩增了几乎完整的16S rRNA基因。随后，该产物被用作第二次聚合酶链式反应的模板，该聚合酶链式反应带有组特异性SRB引物。进行第三轮扩增，获得适合DGGE的片段。用硫化弧菌-硫磺微生物组的特异引物扩增的产物在DGGE图谱中观察到最多的条带，表明这些SRB具有很大的多样性。此外，还检测到了其他系统发育SRB类群的成员，即Desulfotomaculum、Desulfobulbus和Desulfoins-Desulfonema-Desulfosarcina。在生物反应器样品中未检测到与脱硫杆菌和脱硫杆菌相对应的条带。DGGE区带的比较序列分析揭示了群落成员的同一性。开发的三步PCR-DGGE策略是研究硫酸盐还原菌多样性的一个受欢迎的工具。
Here, we describe a three-step nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to detect sulfate-reducing bacteria (SRB) in complex microbial communities from industrial bioreactors. In the first step, the nearly complete 16S rRNA gene was amplified using bacterial primers. Subsequently, this product was used as a template in a second PCR with group-specific SRB primers. A third round of amplification was conducted to obtain fragments suitable for DGGE. The largest number of bands was observed in DGGE patterns of products obtained with primers specific for the Desulfovibrio-Desulfomicrobium group, indicating a large diversity of these SRBs. In addition, members of other phylogenetic SRB groups, i.e., Desulfotomaculum, Desulfobulbus, and Desulfococcus-Desulfonema-Desulfosarcina, were detected. Bands corresponding to Desulfobacterium and Desulfobacter were not detected in the bioreactor samples. Comparative sequence analysis of excised DGGE bands revealed the identity of the community members. The developed three-step PCR-DGGE strategy is a welcome tool for studying the diversity of sulfate-reducing bacteria.