Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0202, USA.
Environ Microbiol. 2010 Apr;12(4):975-89. doi: 10.1111/j.1462-2920.2004.02142.x. Epub 2010 Jan 26.
Heterotrophic bacteria and phytoplankton dominate the biomass and play major roles in the biogeochemical cycles of the surface ocean. Here, we designed and tested a fast, high-throughput and multiplexed hybridization-based assay to detect populations of marine heterotrophic bacteria and phytoplankton based on their small subunit ribosomal DNA sequences. The assay is based on established liquid bead array technology, an approach that is gaining acceptance in biomedical research but remains underutilized in ecology. End-labelled PCR products are hybridized to taxon-specific oligonucleotide probes attached to fluorescently coded beads followed by flow cytometric detection. We used ribosomal DNA environmental clone libraries (a total of 450 clones) and cultured isolates to design and test 26 bacterial and 10 eukaryotic probes specific to various ribotypes and genera of heterotrophic bacteria and eukaryotic phytoplankton. Pure environmental clones or cultures were used as controls and demonstrated specificity of the probes to their target taxa. The quantitative nature of the assay was demonstrated by a significant relationship between the number of target molecules and fluorescence signal. Clone library sequencing and bead array fluorescence from the same sample provided consistent results. We then applied the assay to a 37-day time series of coastal surface seawater samples from the Southern California Bight to examine the temporal dynamics of microbial communities on the scale of days to weeks. As expected, several bacterial phylotypes were positively correlated with total bacterial abundances and chlorophyll a concentrations, but others were negatively correlated. Bacterial taxa belonging to the same broad taxonomic groups did not necessarily correlate with one another, confirming recent results suggesting that inferring ecological role from broad taxonomic identity may not always be accurate.
异养细菌和浮游植物是海洋表面生物量的主要组成部分，在生物地球化学循环中发挥着重要作用。在这里，我们设计并测试了一种快速、高通量和多重基于杂交的检测方法，该方法基于小亚基核糖体 DNA 序列，用于检测海洋异养细菌和浮游植物的种群。该检测方法基于已建立的液珠阵列技术，该方法在生物医学研究中越来越受欢迎，但在生态学中仍未得到充分利用。末端标记的 PCR 产物与特异性寡核苷酸探针杂交，这些探针连接到荧光编码珠上，然后通过流式细胞术检测。我们使用核糖体 DNA 环境克隆文库（总共 450 个克隆）和培养分离物来设计和测试 26 个细菌和 10 个真核探针，这些探针针对各种异养细菌和真核浮游植物的核糖体型和属具有特异性。纯环境克隆或培养物用作对照，并证明了探针对其目标分类群的特异性。通过目标分子数量与荧光信号之间的显著关系，证明了该检测方法的定量性质。来自同一样品的克隆文库测序和珠阵列荧光提供了一致的结果。然后，我们将该检测方法应用于南加州湾沿海表层海水的 37 天时间序列，以检查微生物群落在数天到数周的时间尺度上的动态变化。正如预期的那样，一些细菌类群与总细菌丰度和叶绿素 a 浓度呈正相关，但其他类群呈负相关。属于同一广泛分类群的细菌类群彼此不一定相关，这证实了最近的研究结果表明，从广泛的分类身份推断生态作用并不总是准确的。
