Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces
Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces
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DOI：
10.1128/aem.68.11.5445-5451.2002
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发表时间：
2002-11-01
影响因子：
4.4
通讯作者：
Tanaka, R
中科院分区：
文献类型：
--
作者：
Matsuki, T;Watanabe, K;Tanaka, R
为了检测和鉴定人类粪便中的优势细菌，设计并评估了针对脆弱类杆菌群、双歧杆菌群、球状梭菌群和普雷沃氏菌群的 16S rRNA 基因靶向群特异性引物。这些引物的特异性通过从人类肠道微生物群中常见的 90 个物种中提取的 DNA 得到证实。然后使用组特异性引物从 6 名健康志愿者的粪便中鉴定出 300 个分离株。明确鉴定出脆弱拟杆菌组 117 株、双歧杆菌组 22 株、球状梭菌组 65 株、普雷沃氏菌 17 株，表明 74% 的菌株被 4 对引物鉴定出。其余 79 个分离株通过 16S 核糖体 DNA 序列分析进行鉴定，其中包括 40 个柯林斯氏菌分离株、24 个薄梭菌亚群分离株和 15 个不同簇的分离株。此外，使用从粪便样本中提取的 DNA 无需培养即可完成对这些细菌群的定性检测。这种特定 PCR 技术的目标是开发一种定量检测这些细菌群的方法，目前正在研究用于检测双歧杆菌的实时定量 PCR（T. Requena、J. Burton、T. Matsuki、K. Munro、M. A. Simon、R. Tanaka、K. Watanabe 和 G. W. Tannock，Appl. Environ. Microbiol. 68：2420-2427， 2002）。因此，使用此处描述的组特异性引物检测和识别主要细菌的方法应有助于肠道菌群的组成和动态的未来研究。
For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted group-specific primers for the Bacteroides fragilis group, Bifidobacterium, the Clostridium coccoides group, and Prevotella were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the B. fragilis group, 22 isolates of Bifidobacterium, 65 isolates of the C. coccoides group, and 17 isolates of Prevotella, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of Collinsella, 24 isolates of the Clostridium leptum subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of Bifidobacterium is now being investigated (T. Requena, J. Burton, T. Matsuki, K. Munro, M. A. Simon, R. Tanaka, K. Watanabe, and G. W. Tannock, Appl. Environ. Microbiol. 68:2420-2427, 2002). Therefore, the approaches used to detect and identify predominant bacteria with the group-specific primers described here should contribute to future studies of the composition and dynamics of the intestinal microflora.