Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea
Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea
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DOI：
10.1128/aem.70.8.4971-4979.2004
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发表时间：
2004-08-01
影响因子：
4.4
通讯作者：
Höfle, MG
中科院分区：
文献类型：
--
作者：
Labrenz, M;Brettar, I;Höfle, MG
我们开发了一种高度敏感的方法来评估波罗的海中部水样中未培养细菌的丰度，以一种与反硝化硫微螺旋体相关的未培养的“Epsilonproteobacteria”成员为例。环境海水样品和目标分类单元富集的样品提供了一个独特的机会，可以在广泛的丰度范围内测试该方法。该方法是基于分类群和结构域特异性实时PCR测量的结合，确定相对的T.反硝化菌样16S rRNA基因和16S rRNA丰度，以及总细胞计数和环境RNA含量的测定。它可以量化T.反硝化样16S rRNA分子或16S rRNA基因，并计算每个T.反硝化样细胞的核糖体数量。每个实时测量值及其特异性引物系统使用从原始栖息地获得的环境核酸进行外部标准化校准。这些标准品，以及相应的待测样品，都是由相同的DNA或RNA提取物制备的。富集样品可以直接分析，而环境模板在定量前必须用一般细菌引物预先扩增。预扩增使测定的灵敏度提高了4个数量级以上。有或没有预扩增步骤的富集定量产生了可比的结果。T.反硝化菌样16S rRNA分子的相对丰度为7.1 × 10(3)至4.4 × 10(9)拷贝ml(-1)，相对丰度为0.002至49.7%。T.反硝化菌样16S rRNA基因的相对丰度为9.0 × 10(1) ~ 2.2 × 10(6)拷贝ml(-1)，相对丰度为0.01 ~ 49.7%。实时pcr检测限为20 +/- 16S rRNA分子或0.2 +/- 16S rRNA基因ml（-1）。据估计，在海水中每个T.反硝化样细胞的核糖体数量在20到200之间，在富集中达到2000。结果表明，我们的实时PCR方法可用于测定未培养海洋细菌分类群的细胞丰度和相对丰度，并提供其在自然环境中的活性水平信息。
We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 X 10(3) to 4.4 X 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 X 10(1) to 2.2 X 10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 +/- 16S rRNA molecules or 0.2 +/- 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.