Improved detection of soil microorganisms using fluorescence in situ hybridization (FISH) and catalyzed reporter deposition (CARD-FISH)
Improved detection of soil microorganisms using fluorescence in situ hybridization (FISH) and catalyzed reporter deposition (CARD-FISH)
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DOI：
10.1016/j.soilbio.2008.03.024
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发表时间：
2008-07-01
影响因子：
9.7
通讯作者：
Tippkoetter, Rolf
中科院分区：
文献类型：
--
作者：
Eickhorst, Thilo;Tippkoetter, Rolf
近年来，分子微生物学方法被应用于土壤微生物多样性的研究。荧光原位杂交(FISH)是一种利用荧光标记的寡核苷酸探针对土壤微生物进行特定染色和计数的方法。然而，FISH染色细胞的检测往往受到背景强烈的自发荧光的影响，尤其是在表层土壤样品中。由于酪胺信号放大(TSA)，探针的目标结合部位的荧光强度显著增加。为了评价样品制备和前处理、TSA和检测程序的影响，对6种不同的土壤进行了研究。结果表明，细胞通透性和TSA是改善土壤微生物原位杂交的两个重要因素。粘土含量较高的土壤在聚碳酸酯过滤器上制备比在玻璃片上制备效果更好。与在常规FISH分析中应用单标记寡核苷酸探针相比，使用特定的荧光过滤系统和染料组合大大提高了杂交细胞的检测。因此，卡片鱼染色的细胞适合于使用数字图像分析进行自动计数。然而，DAPI的反染色必须手动分析，因为它受到自体荧光的强烈影响。(C)2008爱思唯尔有限公司。保留所有权利。
In recent years, methods of molecular microbiology have been used for the investigation of soil microbial diversity. Fluorescence in situ hybridization (FISH) represents a method which allows a specific staining and enumeration of soil microorganisms by using fluorescent-labelled oligonucleotide probes. However, the detection of FISH-stained cells is often affected by strong autofluorescence of the background, especially in samples of the top soils.In this study a more efficient FISH-approach coupled with catalyzed reporter deposition (CARD) was adapted to soils. Due to tyramide signal amplification (TSA) the fluorescence intensity has been considerably increased at the target binding site of a probe.Six different soils were investigated to evaluate the effect of sample preparation and pre-treatments, TSA, and the procedure of detection. The results show that both cell permeabilization and TSA are two important factors which improve in situ hybridization of soil microorganisms. Soils with higher clay contents have shown better results when prepared on polycarbonate filters rather than on glass slides. Using specific fluorescence filter systems and dye combinations the detection of hybridized cells was extensively increased compared with the application of monolabelled oligonucleotide probes in regular FISH-analysis. As a result, CARD-FISH-stained cells were suitable for automated counting using digital image analysis. Nevertheless, the counterstain with DAPI had to be analyzed manually as it was strongly affected by autofluorescence. (C) 2008 Elsevier Ltd. All rights reserved.