Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens
Evaluation of enzymatic cell treatments for application of CARD-FISH to methanogens
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DOI：
10.1016/j.mimet.2007.10.006
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发表时间：
2008-01-01
影响因子：
2.2
通讯作者：
Ohashi, Akiyoshi
中科院分区：
文献类型：
--
作者：
Kubota, Kengo;Imachi, Hiroyuki;Ohashi, Akiyoshi
对几种酶促透化方案（利用溶菌酶、蛋白酶K、无色肽酶或重组假脲菌素内肽酶[PeiW]）进行了评价，以应用辣根过氧化物酶标记的寡核苷酸探针和催化报告沉积（CARD-FISH）对产甲烷菌进行原位杂交。在这项研究中，选择了12个产甲烷菌具有典型的细胞表面结构：假胞壁素，表面层，甲烧软骨素和鞘。在测试的处理中，PeiW处理被观察到是最有效的一种，尽管具有鞘的产甲烷菌被不均匀地染色，并且具有甲烷软骨素的产甲烷菌没有被透化。另一方面，溶菌酶、蛋白酶K。和无色肽酶处理无效或引起细胞溶解，导致弱信号或无信号。采用厌氧颗粒污泥样品进一步评价了PeiW处理的适用性。治疗后CARD-FISH法对宫颈糜烂的检出率明显提高。基于本研究所获得的结果，我们提出PeiW处理作为一种新的透化方法CARD-FISH应用于产甲烷菌。(c)2007 Elsevier B. V.保留所有权利。
Several enzymatic permeabilization protocols (utilizing lysozyme, protemase K, achromopeptidase, or recombinant pseudomurein endopeptidase [PeiW]) were evaluated for application of in situ hybridization with horseradish peroxidase-labeled oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) to methanogens. In this study, twelve methanogens were selected that have typical cell surface structures: pseudomurein, surface layer, methanochondroitin and sheath. Among the treatments tested, PeiW treatment was observed to be the most effective one, although methanogens having a sheath were stained heterogeneously and methanogens having methanochondroitin were not permeabilized. On the other hand, lysozyme, protemase K. and achromopeptidase treatments were ineffective or caused cell-lysis, resulting in weak or no signals. Applicability of PeiW treatment was further evaluated using an anaerobic granular sludge sample. The detection rate of Archaea by CARD-FISH increased remarkably after the treatment. Based on the results obtained in this study, we propose PeiW treatment as a novel permeabilization method for CARD-FISH application to methanogens. (c) 2007 Elsevier B.V. All rights reserved.