Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial Populations in human Feces
Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial Populations in human Feces
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DOI：
10.1128/aem.69.2.1251-1262.2003
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发表时间：
2003-02-01
影响因子：
4.4
通讯作者：
Mochizuki, J
中科院分区：
文献类型：
--
作者：
Nagashima, K;Hisada, T;Mochizuki, J
使用核糖体数据库项目网站上的 T-RFLP 分析程序（称为 TAP T-RFLP）构建了用于靶向 16S rRNA 基因的末端限制性片段长度多态性 (T-RFLP) 的新引物-酶组合，并根据经验检验了它们的性能。通过使用荧光标记的516f引物（大肠杆菌位置516至532）和1510r引物（位置1510至1492），从人粪便DNA中扩增出16S rRNA基因。所得扩增产物用RsaI加BfaI或用BslI消化。当对 8 个个体的粪便 DNA 进行 T-RFLP 时，通过 RsaI 和 BfaI 消化检测到 8 个主要操作分类单位 (OTU)，通过 BslI 消化检测到 14 个主要 OTU。 OTU的分布与TAP T-RFLP的计算机模拟结果一致。对个体粪便 DNA 的 T-RFLP 分析给出了特征图谱，而来自同一样本的重复 DNA 制剂之间的 T-RFLP 图谱的变异性很小。与传统的T-RFLP相比，这种新的T-RFLP方法可以根据末端限制性片段长度轻松预测每个OTU对应的肠道细菌群，并且可以通过克隆和测序来鉴定OTU代表的细菌种类。
New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.