Fluorescent oligonucleotide rDNA probes for specific detection of methane oxidising bacteria
Fluorescent oligonucleotide rDNA probes for specific detection of methane oxidising bacteria
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DOI：
10.1111/j.1574-6941.2000.tb00668.x
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发表时间：
2000-01-01
影响因子：
4.2
通讯作者：
Murrell, JC
中科院分区：
文献类型：
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作者：
Bourne, DG;Holmes, AJ;Murrell, JC
针对不同系统发育组的甲烷氧化菌的16 S rRNA的寡核苷酸探针被设计用于原位检测这些生物体。探针，MG-64，专门检测I型甲烷氧化菌。而探针MA-221和MA-621在全细胞杂交中检测II型甲烷氧化菌。还设计了探针Mc 1029，其在全细胞杂交后仅靶向来自甲基球菌属的生物。所有的探针标记的荧光染料Cy 3和杂交的最佳条件进行了测定。突出显示了I型（MG-64）和IT型（MA-621）探针对非甲烷氧化生物的非特异性靶位点。然而，探针用于研究富集文化和环境时，选择性压力有利于甲烷氧化菌的生长超过其他生物体。这些探针的应用被证明在检测I型甲烷氧化菌与MG-64探针在富集培养从河口样品证明甲烷氧化。通过对河口富集培养物的16 S rDNA分子分析证实了I型甲烷氧化菌的检测，所述16 S rDNA分子分析表明16 S rDNA文库中最丰富的细菌克隆类型与甲基杆菌属菌株BB5.1最密切相关，所述甲基杆菌属菌株BB5.1是也从河口环境中分离的I型甲烷氧化菌。(C)2000年，欧洲微生物学会联合会。由Elsevier Science B. V.出版，版权所有。
Oligonucleotide probes targeting the 16S rRNA of distinct phylogenetic groups of methanotrophs were designed for the in situ detection of these organisms. A probe, MG-64, detected specifically type I methanotrophs. while probes MA-221 and MA-621, detected type II methanotrophs in whole cell hybridisations. A probe Mc1029 was also designed which targeted only organisms from the Methylococcus genus after whole cell hybridisations. All probes were labelled with the fluorochrome Cy3 and optimum conditions for hybridisation were determined. Non-specific target sites of the type I (MG-64) and type IT (MA-621) probes to non-methanotrophic organisms are highlighted. The probes are however used in studying enrichment cultures and environments when selective pressure favours the growth of methanotrophs over other organisms. The application of these probes was demonstrated in the detection of type I methanotrophs with the MG-64 probe in an enrichment culture from an estuarine sample demonstrating methane oxidation. The detection of type I methanotrophs was confirmed by a 16S rDNA molecular analysis of the estuarine enrichment culture which demonstrated that the most abundant bacterial clone type in the 16S rDNA library was most closely related to Methylobacter sp. strain BB5.1, a type I methanotroph also isolated from an estuarine environment. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.