Detection of Actinobacteria cultivated from environmental samples reveals bias in universal primers
Detection of Actinobacteria cultivated from environmental samples reveals bias in universal primers
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DOI：
10.1111/j.1472-765x.2007.02198.x
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发表时间：
2007-10-01
影响因子：
2.4
通讯作者：
Olson, J. B.
中科院分区：
文献类型：
--
作者：
Farris, M. H.;Olson, J. B.
目的：建立放线菌培养培养基，利用放线菌特异性引物对培养的放线菌进行筛选，并对通用引物对放线菌分离物的检测效果进行检验。方法与结果：研制了一种适用于陆崖环境的土壤提取培养基。利用分类特异引物进行聚合酶链反应（PCR）鉴定放线菌。使用通用细菌引物24f和1492r（修改版本和原始版本）扩增推测的放线菌的16S rRNA基因。虽然这两种反向引物都不能提供20% ~ 50%的扩增产物，但1492r引物比1492r-mod更有效地检测到放线菌。15株扩增失败的1492r引物含有退火位点的基因区域与原始1492r引物的核苷酸序列没有差异。结论：通用的16S rRNA基因引物不能从环境样本中的所有细菌中扩增该基因。一些放线菌可能与通用引物具有100%的序列相似性，但仍未被检测到。研究的意义和影响：这些发现对环境样本中特定分类群的研究具有重要意义，在这些环境样本中，使用通用引物的反应可能无法揭示其存在和多样性的程度。
Aims: The aims of this study were to develop media to cultivate actinomycetes, screen the resulting isolates with Actinobacteria-specific primers, and examine the efficacy of detection of the actinobacterial isolates with universal primers.Methods and Results: Soil-extract medium was developed for a terrestrial bluff environment. Recovered isolates were subjected to polymerase chain reaction (PCR) with taxon-specific primers to identify Actinobacteria. Universal bacterial primers 24f and 1492r (modified and original versions) were used to amplify the 16S rRNA gene from the putative Actinobacteria. While both reverse primers failed to provide amplification products from 20% to 50% of the isolates, the 1492r primer detected Actinobacteria more effectively than 1492r-mod. The region of the gene containing the annealing site for the 1492r primers from 15 isolates that failed to amplify showed no differences in nucleotide sequence to the original 1492r primer.Conclusion: Universal 16S rRNA gene primers are not capable of amplifying this gene from all bacteria within an environmental sample. Some Actinobacteria may share 100% sequence similarity to universal primers but remain undetected.Significance and Impact of the study: These findings are important for studies of particular taxa in environmental samples where reactions utilizing universal primers may not reveal the extent of their presence and diversity.