Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes
Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes
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DOI：
10.1016/j.mimet.2011.11.014
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发表时间：
2012-02-01
影响因子：
2.2
通讯作者：
Kubota, Kengo
中科院分区：
文献类型：
--
作者：
Kawakami, Shuji;Hasegawa, Takuya;Kubota, Kengo
单细胞分辨率的功能基因的原位检测是目前微生物学家感兴趣的。在这里，我们开发了一个两次通过酪胺信号放大（TSA）-荧光原位杂交（FISH）协议与PCR衍生的多核苷酸探针检测原核细胞中的单拷贝基因。分别针对产甲烷菌和硫酸盐还原菌中的mcrA基因和apsA基因。该方案显示出明亮的荧光，具有良好的信噪比，并实现了高的检测效率（>98%）。区分阈值约为82-89%序列同一性。利用多核苷酸探针的两次TSA-FISH技术成功地检测了厌氧污泥样品中具有mcrA或apsA基因的微生物。所开发的协议是有用的，用于识别基于功能基因序列的单个微生物细胞。(C)2011爱思唯尔有限公司版权所有。
In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The mcrA gene and the apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (>98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the mcrA or apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences. (C) 2011 Elsevier B.V. All rights reserved.