OPTIMIZING FLUORESCENT INSITU HYBRIDIZATION WITH RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBES FOR FLOW CYTOMETRIC IDENTIFICATION OF MICROORGANISMS
OPTIMIZING FLUORESCENT INSITU HYBRIDIZATION WITH RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBES FOR FLOW CYTOMETRIC IDENTIFICATION OF MICROORGANISMS
复制标题
DOI：
10.1002/cyto.990140205
复制
发表时间：
1993-01-01
期刊：
影响因子：
--
通讯作者：
BEISKER, W
中科院分区：
文献类型：
--
作者：
WALLNER, G;AMANN, R;BEISKER, W
荧光rRNA靶向寡核苷酸探针（“系统发育染色”）和流式细胞术的组合用于混合微生物种群的高分辨率自动分析。固定的细菌和酵母细胞在悬浮液中与荧光素或四甲基罗丹明标记的寡核苷酸探针杂交，所述寡核苷酸探针与16 S核糖体RNA（rRNA）分子的组特异性区域互补。通过流式细胞术定量探针赋予的细胞荧光，我们可以区分靶细胞群和非靶细胞群。我们严格审查的杂交条件，动力学的杂交，和posthybridization治疗的变化。发现中间探针浓度、向杂交缓冲液中添加去污剂和杂交后洗涤步骤可增加信噪比。我们可以证明大肠杆菌和洋葱假单胞菌细胞的生长速率和探针赋予的荧光之间的线性相关性。用多种荧光染料标记的寡核苷酸显示非特异性结合水平升高，因此不能用于降低检测限，这仍然限制了荧光rRNA靶向的研究。 将寡核苷酸探针用于生长良好的微生物细胞。两种不同特异性的探针-一种标记有荧光素，另一种标记有四甲基罗丹明-可以同时应用于双色分析。
A combination of fluorescent rRNA-targeted oligonucleotide probes (''phylogenetic stains'') and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein- or tetramethylrhodamine-labeled oligonucleotide probes complementary to group-specific regions of the 16S ribosomal RNA (rRNA) molecules. Quantifying probe-conferred cell fluorescence by flow cytometry, we could discriminate between target and nontarget cell populations. We critically examined changes of the hybridization conditions, kinetics of the hybridization, and posthybridization treatments. Intermediate probe concentrations, addition of detergent to the hybridization buffer, and a posthybridization washing step were found to increase the signal to noise ratio. We could demonstrate a linear correlation between growth rate and probe-conferred fluorescence of Escherichia coli and Pseudomonas cepacia cells. Oligonucleotides labeled with multiple fluorochromes showed elevated levels of non-specific binding and therefore could not be used to lower the detection limits, which still restrict studies with fluorescing rRNA-targeted oligonucleotide probes to well-growing microbial cells. Two probes of different specificities-one labeled with fluorescein, the other with tetramethylrhodamine-could be applied simultaneously for dual color analysis.