ERIC-PCR fingerprinting-based community DNA hybridization to pinpoint genome-specific fragments as molecular markers to identify and track populations common to healthy human guts
ERIC-PCR fingerprinting-based community DNA hybridization to pinpoint genome-specific fragments as molecular markers to identify and track populations common to healthy human guts
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DOI：
10.1016/j.mimet.2004.06.007
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发表时间：
2004-10-01
影响因子：
2.2
通讯作者：
Zhao, LP
中科院分区：
文献类型：
--
作者：
Wei, GF;Pan, L;Zhao, LP
健康人体肠道常见的细菌种群可能在人类健康中发挥重要作用。利用肠杆菌重复基因间共有序列（ERIC）-PCR指纹图谱，开发了一种发现基因组序列作为这些细菌标记的新策略。微生物群落内的结构特征与ERIC-PCR进行比较，然后进行DNA杂交，以鉴定来自健康人类个体的样品所共有的基因组片段。来自12个患病或健康的人类和仔猪受试者的粪便样品的ERIC-PCR图谱显示每个受试个体的稳定、独特的带型。通过在高严格条件下与来自一名健康儿童粪便样品的DIG标记的ERIC-PCR产物杂交，检查样品之间相同大小条带中DNA片段的序列同源性。与原始琼脂糖指纹的杂交图谱的比较分析确定了三个主要的带作为与健康人肠道相关的群体的签名，大小为500，800和1000 bp。三个条带的克隆文库分析产生了17个基因组片段，其中三个仅与多形拟杆菌基因组的区域显示出高相似性，而其余的是孤儿序列。通过序列选择性PCR实验验证了这些序列与健康肠道的关联，该实验表明，在所有32名健康人和13只健康仔猪中均存在单一片段。两个片段存在于健康人组和18名非感染性腹泻儿童中，但不存在于8名感染性腹泻儿童中。用这种新策略鉴定的基因组片段可用作基因组特异性标记物，用于动态监测和序列指导分离复杂群落中功能重要的细菌群体，如人类肠道微生物群落。(C)2004 Elsevier B. V.保留所有权利。
Bacterial populations common to healthy human guts may play important roles in human health. A new strategy for discovering genomic sequences as markers for these bacteria was developed using Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR fingerprinting. Structural features within microbial communities are compared with ERIC-PCR followed by DNA hybridization to identify genomic fragments shared by samples from healthy human individuals. ERIC-PCR profiles of fecal samples from 12 diseased or healthy human and piglet subjects demonstrated stable, unique banding patterns for each individual tested. Sequence homology of DNA fragments in bands of identical size was examined between samples by hybridization under high stringency conditions with DIG-labeled ERIC-PCR products derived from the fecal sample of one healthy child. Comparative analysis of the hybridization profiles with the original agarose fingerprints identified three predominant bands as signatures for populations associated with healthy human guts with sizes of 500, 800 and 1000 bp. Clone library profiling of the three bands produced 17 genome fragments, three of which showed high similarity only with regions of the Bacteroides thetaiotaomicron genome, while the remainder were orphan sequences. Association of these sequences with healthy guts was validated by sequence-selective PCR experiments, which showed that a single fragment was present in all 32 healthy humans and 13 healthy piglets tested. Two fragments were present in the healthy human group and in 18 children with non-infectious diarrhea but not in eight children with infectious diarrhea. Genome fragments identified with this novel strategy may be used as genome-specific markers for dynamic monitoring and sequence-guided isolation of functionally important bacterial populations in complex communities such as human gut microflora. (C) 2004 Elsevier B.V. All rights reserved.