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首页> 外文期刊>Systematic and Applied Microbiology >Evaluation of the environmental specificity of Fluorescence In Situ Hybridization (FISH) using Fluorescence-Activated Cell Sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil. (Special Issue: Fluorescence In Situ Hybridization (FISH).)
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Evaluation of the environmental specificity of Fluorescence In Situ Hybridization (FISH) using Fluorescence-Activated Cell Sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil. (Special Issue: Fluorescence In Situ Hybridization (FISH).)

机译：使用从根际土壤中提取的探针（PSE1284）阳性细胞的荧光激活细胞分选（FACS），评估荧光原位杂交（FISH）的环境特异性。 （特刊：荧光原位杂交（FISH）。）

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We explicitly tested for the first time the 'environmental specificity' of traditional 16S rRNA-targeted Fluorescence In Situ Hybridization (FISH) through comparison of the bacterial diversity actually targeted in the environment with the diversity that should be exactly targeted (i.e. without mismatches) according to in silico analysis. To do this, we exploited advances in modern Flow Cytometry that enabled improved detection and therefore sorting of sub-micron-sized particles and used probe PSE1284 (designed to target Pseudomonads) applied to Lolium perenne rhizosphere soil as our test system. The 6-carboxyfluorescein (6-FAM)-PSE1284-hybridized population, defined as displaying enhanced green fluorescence in Flow Cytometry, represented 3.51+or-1.28% of the total detected population when corrected using a nonsense (NON-EUB338) probe control. Analysis of 16S rRNA gene libraries constructed from Fluorescence Activated Cell Sorted-recovered fluorescent populations (n=3), revealed that 98.5% (Pseudomonas spp. comprised 68.7% and Burkholderia spp. 29.8%) of the total sorted population was specifically targeted as evidenced by the homology of the 16S rRNA sequences to the probe sequence. In silico evaluation of probe PSE1284 with the use of RDP-10 probeMatch justified the existence of Burkholderia spp. among the sorted cells. The lack of novelty in Pseudomonas spp. sequences uncovered was notable, probably reflecting the well-studied nature of this functionally important genus. To judge the diversity recorded within the FACS-sorted population, rarefaction and DGGE analysis were used to evaluate, respectively, the proportion of Pseudomonas diversity uncovered by the sequencing effort and the representativeness of the NycodenzReg. method for the extraction of bacterial cells from soil.Digital Object Identifier http://dx.doi.org/10.1016/j.syapm.2011.11.009
机译：通过比较实际靶向环境中的细菌多样性与应准确靶向的多样性（即无错配），我们首次明确测试了传统的16S rRNA靶向荧光原位杂交（FISH）的“环境特异性”。进行计算机分析。为此，我们利用了现代流式细胞仪的先进技术，该技术能够改进检测并因此对亚微米级颗粒进行分类，并使用应用于黑麦草根际土壤的探针PSE1284（旨在针对假单胞菌）作为我们的测试系统。 6-羧基荧光素（6-FAM）-PSE1284杂化的群体在流式细胞仪中定义为显示增强的绿色荧光，当使用无义（NON-EUB338）探针对照进行校正时，代表检测到的总群体的3.51％或-1.28％。分析从荧光激活细胞分选回收的荧光种群（n = 3）构建的16S rRNA基因文库，发现有针对性地确定了总分选种群的98.5％（假单胞菌属占68.7％，伯克霍尔德氏菌属占29.8％）是有针对性的。 16S rRNA序列与探针序列的同源性。使用RDP-10 probeMatch对PSE1284探针进行的计算机评估，证明了Burkholderia spp的存在。在排序的单元格中。假单胞菌属物种缺乏新颖性。发现的序列是值得注意的，可能反映了该功能重要属的充分研究的性质。为了判断在FACS分类种群中记录的多样性，分别使用稀疏度和DGGE分析来评估测序工作和NycodenzReg的代表性所揭示的假单胞菌多样性的比例。从土壤中提取细菌细胞的方法。数字对象标识符http://dx.doi.org/10.1016/j.syapm.2011.11.009

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