Identification of bacterial diversity in the oyster Crassostrea gigas by fluorescent in situ hybridization and polymerase chain reaction
Identification of bacterial diversity in the oyster Crassostrea gigas by fluorescent in situ hybridization and polymerase chain reaction
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DOI：
10.1111/j.1365-2672.2005.02800.x
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发表时间：
2006-04-01
影响因子：
4
通讯作者：
Olmos-Soto, J
中科院分区：
文献类型：
--
作者：
Hernández-Zárate, G;Olmos-Soto, J
目的：为了对墨西哥托多斯桑托斯湾长牡蛎（Crassostreagigas）的细菌多样性进行快速、可靠的鉴定，本研究应用荧光原位杂交（FISH）和聚合酶链反应（PCR）技术对长牡蛎的细菌多样性进行了鉴定。为了达到这一目标，属和组特异性的寡核苷酸针对16 S rDNA/RRNAwere used.Methods and Results：牡蛎收集和不同的组织进行了分析，通过培养独立的方法。在消化腺和生殖腺γ-变形菌和革兰氏阳性菌与低G+C含量，被确定为代谢活跃的FISH。在牡蛎鳃中观察到更高的活性多样性，包括具有低和高G+C含量的革兰氏阳性细菌，噬纤维菌/黄杆菌属簇和γ-变形菌门的成员。与FISH分析一致，用属和组特异性寡核苷酸扩增16 S rDNA基因片段证实了相同组的存在，以及α-和β-变形杆菌属、假单胞菌属的成员。和芽孢杆菌属。结论：准确和易于应用的分子方法相结合，使我们能够快速筛选牡蛎中的高细菌多样性。这项工作是第一个报告的细菌多样性分析牡蛎组织FISH和PCR，而不使用依赖于培养的方法，并允许我们确定细菌群落的系统发育多样性存在于牡蛎文化，包括具有和不具有代谢活性的细菌，以及不可培养的细胞，这些细胞通常被传统鉴定低估。
Aims: To carry out a rapid and reliable identification of bacterial diversity in the oyster Crassostrea gigas from Todos Santos Bay, Mexico, in the current study we applied the molecular techniques of fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). In order to reach this goal, genus and group-specific oligonucleotides targeted to 16S rDNA/rRNA were used.Methods and Results: Oysters were collected and different tissues were analysed by means of culture-independent methodologies. In the digestive glands and gonads gamma-Proteobacteria and Gram-positive bacteria with a low G+C content, were identified as metabolically active by FISH. In the oyster gills a higher active diversity was observed, including Gram-positive bacteria with a low and high G+C content, members of the Cytophaga/Flavobacterium cluster and gamma-Proteobacteria. Consistent with FISH analysis, the amplification of 16S rDNA genes fragments with genus and group-specific oligonucleotides confirmed the presence of the same groups, as well as members of the alpha- and beta-Proteobacterias, Pseudomonas spp. and Bacillus spp.Conclusions: The combination of accurate and very easy-to-apply molecular methods allowed us to carry out a rapid screening of high bacterial diversity in oysters.Significance and Impact of the Study: This work is the first report about bacterial diversity in oyster tissues analysed by FISH and PCR, without using culture-dependent methods and allowed us to determine the phylogenetic diversity of the bacterial communities present in oyster cultures, including bacteria with and without metabolic activity, as well as uncultivable cells, which are generally underestimated by traditional identification.