Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH
Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH
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通过荧光原位杂交 (FISH) 和催化报告基因沉积 -FISH 对检测微生物所需的目标分子进行定量
DOI：
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发表时间：
2008
影响因子：
4.4
通讯作者：
M. Wagner
中科院分区：
文献类型：
--
作者：
T. Hoshino;L. S. Yilmaz;D. Noguera;H. Daims;M. Wagner
摘要以rRNA为靶点的寡核苷酸探针荧光原位杂交(FISH)是一种利用荧光显微镜对环境样品和医学样品中的微生物进行检测和定量的方法。如果探测目标生物的rRNA含量低，导致背景荧光检测不到的微弱荧光信号，鱼类就会出现困难。这一局限性通过诸如催化报告沉积(CARD)-FISH等技术修改而得到改善，但在FISH或CARD-FISH之后获得微生物细胞的可见信号所需的最小rRNA拷贝数以前尚未确定。本研究发展了一种新的竞争性FISH方法，并基于探针竞争的热力学模型，在混合纯培养物和活性污泥中用FISH和CARD-FISH检测细菌所需的每细胞16S rRNA拷贝数。用Cy3标记的探针EUB338-I进行常规FISH的检测下限为370±45个16S rRNA分子/细胞和活性污泥中16S rRNA拷贝数1400±170个/细胞。CARD-FISH的灵敏度分别为8.9±1.5~14±2和36±6~54±7个16S rRNA分子/细胞，表明CARD-FISH的灵敏度是常规FISH的26~41倍。这些结果表明，使用寡核苷酸探针的优化FISH方法可能适用于FISH的更新应用(例如，原位检测微生物细胞中的mRNA)。
ABSTRACT Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).
影响因子：
56.9
作者：
DELONG, EF;WICKHAM, GS;PACE, NR
通讯作者：
PACE, NR