Sequence-Specific Cleavage of Small-Subunit (SSU) rRNA with Oligonucleotides and RNase H: a Rapid and Simple Approach to SSU rRNA-Based Quantitative Detection of Microorganisms
Sequence-Specific Cleavage of Small-Subunit (SSU) rRNA with Oligonucleotides and RNase H: a Rapid and Simple Approach to SSU rRNA-Based Quantitative Detection of Microorganisms
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DOI：
10.1128/aem.70.6.3650-3663.2004
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发表时间：
2004-06
影响因子：
4.4
通讯作者：
Y. Uyeno;Y. Sekiguchi;A. Sunaga;H. Yoshida;Y. Kamagata
中科院分区：
文献类型：
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作者：
Y. Uyeno;Y. Sekiguchi;A. Sunaga;H. Yoshida;Y. Kamagata
开发了一种基于小亚单位（SSU） rrna的复杂生态系统中特定微生物群定量检测的快速简便方法。该方法使用寡核苷酸和RNase H对rRNA分子进行序列特异性切割。将确定的SSU rRNA混合物与寡核苷酸（称为“剪刀探针”）混合，该寡核苷酸专门设计用于与目标rRNA的特定位点杂交，随后用RNase H进行酶切，在杂交位点进行序列依赖性rRNA切割。在适当的反应条件下，靶向rnas被正确地切割成两个片段，而非靶向rnas在相同的条件下保持完整。通过控制rRNA与寡核苷酸之间的杂交强度，即通过控制反应温度或用于反应的杂交消化缓冲液中甲酰胺的浓度，可以适当地调节裂解的特异性。这使得完全匹配的rRNA序列与单碱基不匹配的序列能够可靠地区分。为了检测目标rRNA，用核苷酸染色荧光染料凝胶电泳分析得到的RNA片段模式，以分离裂解和完整的rRNA分子。通过测定电泳图中单个SSU rRNA条带的信号强度，可以很容易地计算出目标SSU rRNA片段在总SSU rRNA中的相对丰度，而无需使用外部标准。该方法提供了一种基于rRNA的临床和环境标本中特定微生物群的快速简便的鉴定、检测和定量方法。
ABSTRACT A rapid and simple approach to the small-subunit (SSU) rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems has been developed. The method employs sequence-specific cleavage of rRNA molecules with oligonucleotides and RNase H. Defined mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to as a “scissor probe”) that was specifically designed to hybridize with a particular site of targeted rRNA and were subsequently digested with RNase H to proceed to sequence-dependent rRNA scission at the hybridization site. Under appropriate reaction conditions, the targeted rRNAs were correctly cut into two fragments, whereas nontargeted rRNAs remained intact under the same conditions. The specificity of the cleavage could be properly adjusted by controlling the hybridization stringency between the rRNA and the oligonucleotides, i.e., by controlling either the temperature of the reaction or the formamide concentration in the hybridization-digestion buffer used for the reaction. This enabled the reliable discrimination of completely matched rRNA sequences from single-base mismatched sequences. For the detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel electrophoresis with nucleotide-staining fluorescent dyes in order to separate cleaved and intact rRNA molecules. The relative abundance of the targeted SSU rRNA fragments in the total SSU rRNA could easily be calculated without the use of an external standard by determining the signal intensity of individual SSU rRNA bands in the electropherogram. This approach provides a fast and easy means of identification, detection, and quantification of a particular group of microbes in clinical and environmental specimens based on rRNA.