Analysis of Oral Microbiota in Children with Dental Caries by PCR-DGGE and Barcoded Pyrosequencing
Analysis of Oral Microbiota in Children with Dental Caries by PCR-DGGE and Barcoded Pyrosequencing
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DOI：
10.1007/s00248-010-9712-8
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发表时间：
2010-10-01
影响因子：
3.6
通讯作者：
Xiang, Charlie
中科院分区：
文献类型：
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作者：
Ling, Zongxin;Kong, Jianming;Xiang, Charlie
口腔微生物群在维持口腔内环境稳定方面起着至关重要的作用。龋齿是儿童最常见的口腔疾病之一，致病菌有助于疾病的发展。然而，迄今为止，龋齿儿童口腔中细菌群落的整体结构尚未得到深入研究。我们使用高通量条形码焦磷酸测序和PCR-变性梯度凝胶电泳（DGGE）来检测来自中国的60名3至6岁患有和不患有龋齿的儿童的唾液和龈上菌斑中口腔微生物群的细菌多样性。多重条形码焦磷酸测序在单次运行中进行，其中多个样品由多重标识符唯一标记。由于PCR-DGGE分析是一种传统的分子生态学方法，因此也对相同的样品进行了这种分析，并比较了两种方法的结果。共获得186，787个高质量序列用于评估细菌多样性，其中41，905个独特序列代表所有细菌类型。我们发现，儿童口腔微生物群的多样性远远超过以往的研究报告，在口腔中发现了属于10门的200多个属。唾液和龈上菌斑中的细菌类型有显著性差异，可分为两个明显的类群（p < 0.05）。通过PCR-DGGE和条形码焦磷酸测序分析口腔微生物组中的细菌多样性来交叉验证数据集。菌斑中的链球菌属、韦荣氏球菌属、放线菌属、颗粒菌属、纤毛菌属和硫单胞菌属与龋病发生有显著相关性（p < 0.05）。结果表明，菌斑中不存在单一的致病菌，而存在与龋病密切相关的致病菌群。口腔微生物群的巨大多样性使我们能够更好地了解口腔微生态系统，这些菌斑中的致病菌群为龋齿的病因学提供了新的见解，并为疾病的干预提供了新的目标。
Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p < 0.05). The bacterial diversity in oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p < 0.05). The results showed that there was no one specific pathogen but rather pathogenic populations in plaque that significantly correlated with dental caries. The enormous diversity of oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.