A DNA microarray platform based on direct detection of rRNA for characterization of freshwater sediment-related prokaryotic communities
A DNA microarray platform based on direct detection of rRNA for characterization of freshwater sediment-related prokaryotic communities
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DOI：
10.1128/aem.02949-05
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发表时间：
2006-07-01
影响因子：
4.4
通讯作者：
Manz, Werner
中科院分区：
文献类型：
--
作者：
Peplies, Joerg;Lachmund, Christine;Manz, Werner
基于70个16S rrna靶向寡核苷酸探针和直接标记的环境RNA，开发并评估了用于表征淡水沉积物细菌群落的DNA微阵列平台。应用一种简单的协议，对氨基硅烷包被的载玻片进行有效的背景阻断，提高了信噪比，对特定的16S rRNA靶标的检测限为10 ng。使用来自不同系统发育谱系的纯培养物的RNA对该系统进行了初步特异性测试，结果显示，经过方案优化后，假阳性信号的比例接近5%，正确阳性信号的边际损失。随后对来自四个不同德国河流遗址的沉积物相关群落RNA的微阵列分析表明，目标群体的多样性较低，但群落组成存在明显差异。平行荧光原位杂交结合敏感催化报告细胞沉积（CARD-FISH）技术支持了这一结果。在不同采样点的数据比较中，建议对相对细胞丰度低至2%的群体进行特定检测，并建议将微阵列信号强度与群体大小相关联。我们的研究结果表明，通过使用标准的单细胞杂交探针和直接检测环境rRNA， DNA微阵列技术可以快速有效地预表征复杂的细菌群落，也可以在方法上具有挑战性的栖息地，如异质性的淡水沉积物中进行。
A DNA microarray platform for the characterization of bacterial communities in freshwater sediments based on a heterogeneous set of 70 16S rRNA-targeted oligonucleotide probes and directly labeled environmental RNA was developed and evaluated. Application of a simple protocol for the efficient background blocking of aminosilane-coated slides resulted in an improved signal-to-noise ratio and a detection limit of 10 ng for particular 16S rRNA targets. An initial specificity test of the system using RNA from pure cultures of different phylogenetic lineages showed a fraction of false-positive signals of similar to 5% after protocol optimization and a marginal loss of correct positive signals. Subsequent microarray analysis of sediment-related community RNA from four different German river sites suggested low diversity for the groups targeted but indicated distinct differences in community composition. The results were supported by parallel fluorescence in situ hybridization in combination with sensitive catalyzed reporter deposition (CARD-FISH). In comparisons of the data of different sampling sites, specific detection of populations with relative cellular abundances down to 2% as well as a correlation of microarray signal intensities and population size is suggested. Our results demonstrate that DNA microarray technology allows for the fast and efficient precharacterization of complex bacterial communities by the use of standard single-cell hybridization probes and the direct detection of environmental rRNA, also in methodological challenging habitats such as heterogeneous lotic freshwater sediments.