Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets
Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets
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DOI：
10.1111/j.1399-302x.2005.00255.x
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发表时间：
2006-02-01
影响因子：
--
通讯作者：
Wade, WG
中科院分区：
文献类型：
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作者：
de Lillo, A;Ashley, FP;Wade, WG
简介：基于 16S rRNA 基因序列分析的分子生态分析已广泛应用于复杂细菌群落的表征。然而，由于引物和目标生物序列之间的不匹配，“通用”PCR 引物可能会给克隆文库的物种组成分析带来偏差。在本研究中，比较了三种通用引物组，以分析龈下菌斑中的微生物群落。方法：从两名患有局部严重慢性牙周炎的受试者中收集了三个龈下菌斑样本。每个样品的一半进行培养，同时从剩余的一半中提取 DNA，并使用通用引物对 27F、1525R 扩增 16S rDNA (A)； 27F、1492R（B）和 530F、1525R（C）。对扩增的基因进行克隆、测序和鉴定，并与16S rRNA数据库进行比对。结果：177个分离株中鉴定出137个类群，已测序417个克隆。其中，86 个仅通过分子技术检测到，而 26 个仅通过培养发现。 81 个分类单元的序列与指定物种的序列不对应，其中 38 个分类单元未在核苷酸数据库中表示。对这38个分类群的16S RNA基因进行了测序并存入GenBank。结论：使用三组通用引物可以鉴定出38个新的细菌系统型。不同引物组生成的文库组成存在显着差异。建议结合分子和培养技术来最大限度地扩大口腔样本中细菌分类群的检测范围。
Introduction: Molecular ecological analysis based on 16S rRNA gene sequence analysis is well established for the characterisation of complex bacterial communities. However, 'universal' PCR primers can introduce biases into the analysis of the species composition of clone libraries because of mismatches between the primer and target organism sequences. In this study, three universal primer sets were compared for the analysis of the microflora in subgingival plaque.Methods: Three subgingival plaque samples were collected from two subjects with localised severe chronic periodontitis. Half of each sample was cultured while DNA was extracted from the remaining half and 16S rDNA amplified with universal primer pairs 27F, 1525R (A); 27F, 1492R (B) and 530F, 1525R (C). Amplified genes were cloned, sequenced and identified by comparison with 16S rRNA databases.Results: 137 taxa were identified among 177 isolates and 417 clones sequenced. Of these, 86 were detected only by the molecular technique whereas 26 were found only by culture. Sequences from 81 taxa did not correspond to those of named species and of these, 38 were not represented in the nucleotide databases. 16S RNA genes for these 38 taxa were sequenced and deposited with GenBank.Conclusion: The use of three sets of universal primers allowed the identification of 38 novel bacterial phylotypes. There were marked differences in the composition of the libraries generated by the different primer sets. A combination of molecular and cultural techniques is recommended to maximise the coverage of detection of bacterial taxa in oral samples.