Sequence-specific cleavage of 16S rRNA for rapid and quantitative detection of particular groups of anaerobes in bioreactors
Sequence-specific cleavage of 16S rRNA for rapid and quantitative detection of particular groups of anaerobes in bioreactors
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DOI：
10.2166/wst.2005.0505
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发表时间：
2005-01-01
影响因子：
2.7
通讯作者：
Kamagata, Y
中科院分区：
文献类型：
--
作者：
Sekiguchi, Y;Uyeno, Y;Kamagata, Y
我们开发了一种快速简便的方法，用于复杂生态系统中特定微生物的定量检测。该方法依赖于用核糖核酸酶H（RNase H）和与靶向rRNA分子特异性杂交的寡核苷酸对16S rRNA进行序列特异性切割。首先将来自复杂群落的RNA与寡核苷酸混合，随后用RNase H消化以在杂交位点实现序列依赖性rRNA切割。对于靶向rRNA的定量检测，通过凝胶电泳分析所得RNA片段模式，其分离并定量切割的和完整的rRNA片段。该方法能够通过相对简单和快速的实验程序定量检测复杂微生物群落中的微生物。然后，我们将裂解方法应用于实际的厌氧微生物群落，如消化污水污泥和UASB污泥。结果表明，该方法完全适用于含有复杂厌氧微生物的厌氧生物反应器生态系统。
We developed a rapid and simple method for rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems. The method relies on the sequence-specific scission of 16S rRNA with ribonuclease H (RNase H) and oligonucleotides that specifically hybridize with targeted rRNA molecules. RNAs from a complex community were first mixed with an oligonucleotide and were subsequently digested with RNase H to achieve sequence-dependent rRNA cleavage at the hybridization site. For the quantitative detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel-electrophoresis, which separated and quantified cleaved and intact rRNA fragments. This method enabled the quantitative detection of microbes in a complex microbial community by a relatively simple and fast experimental procedure. We then applied the cleavage method to actual anaerobic microbial communities such as digested sewage sludge and UASB sludges. The results demonstrated that the present method was fully applicable to anaerobic digestor ecosystems containing complex anaerobic microorganisms.