Molecular method to assess the diversity of Burkholderia species in environmental samples
Molecular method to assess the diversity of Burkholderia species in environmental samples
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DOI：
10.1128/aem.68.4.1595-1603.2002
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发表时间：
2002-04-01
影响因子：
4.4
通讯作者：
van Elsas, JD
中科院分区：
文献类型：
--
作者：
Salles, JF;De Souza, FA;van Elsas, JD
尽管伯克霍尔德菌属的许多成员在土壤微生物群落中的重要性，但迄今为止还没有直接的方法来评估该属的多样性。基于土壤dna的pcr -变性梯度电泳（DGGE）技术是研究土壤微生物群落多样性的有力工具，可用于土壤样品中伯克霍尔德菌的多样性检测和分析。基于16S rRNA基因序列开发了伯克霍尔德菌属特异性引物，并以伯克霍尔德菌属和非伯克霍尔德菌属的基因组dna为模板进行pcr评价。所使用的引物系统对大多数已建立的伯克霍尔德菌属物种具有良好的特异性和敏感性。对PCR产物的DGGE分析表明，在迁移行为上存在足够的差异，可以区分14种伯克霍尔德菌中的大多数。对土壤DNA扩增子进行序列分析，发现与伯克霍尔德菌属相关的序列，表明PCR-DGGE方法适用于自然环境下伯克霍尔德菌属多样性的研究。PCR-DGGE分析显示，2个草地样地伯克霍尔德菌群落多样性差异主要存在于根际土壤样品和块状土壤样品之间；后一个样本中的群落产生了更复杂的模式。
In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient get electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.