16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales"
16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales"
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DOI：
10.1128/aem.71.3.1373-1386.2005
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发表时间：
2005-03-01
影响因子：
4.4
通讯作者：
Wagner, M
中科院分区：
文献类型：
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作者：
Loy, A;Schulz, C;Wagner, M
为了同时鉴定环境样品中的β-变形菌目“红环菌目”的成员，开发了由79个探针组成的16 S rRNA基因靶向寡核苷酸芯片（RHC-PhyloChip）。探针设计基于来自所有培养的和尚未培养的“红环菌目”成员的可用16 S rRNA序列的系统发育分析。“多重巢式探针组与来自29种参考生物的16 S rRNA基因PCR扩增子进行了微阵列杂交评估。随后，RHC-PhyloChip被成功地用于工业废水处理厂活性污泥中不依赖培养的“红环藻目”多样性分析。一个新设计的“红环菌”选择性PCR扩增系统的微阵列杂交前的实施大大提高了灵敏度的RHC-PhyloChip，从而使检测“红环菌”种群的相对丰度小于1%的所有细菌（荧光原位杂交测定）在活性污泥中。尚未培养的动胶菌属，铁杆菌属/脱氯单胞菌属，和Sterolibacterium相关的细菌在工业活性污泥中的存在下，由RHC-PhyloChip分析表明，证实了其16 S rRNA基因序列的检索和随后的系统发育分析，证明了RHC-PhyloChip作为一种新的环境微生物学监测工具的适用性。
For simultaneous identification of members of the betaproteobacterial order "Rhodocyclales" in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the "Rhodocyclales." The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent "Rhodocyclales" diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed "Rhodocyclales"-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of "Rhodocyclales" populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.