Key Laboratory of Ministry of Education for Microbial Metabolism, School of Life Science & Biotechnology, Shanghai Jiao Tong University, China.
Arch Microbiol. 2011 May;193(5):341-50. doi: 10.1007/s00203-011-0679-8. Epub 2011 Jan 28.
DNA restriction fragment polymorphism technologies such as amplified ribosomal DNA restriction analysis (ARDRA) and terminal restriction fragment length polymorphism (T-RFLP) have been widely used in investigating microbial community structures. However, these methods are limited due to either the low resolution or sensitivity. In this study, a fluorophore-ribosomal DNA restriction typing (f-DRT) approach is developed for structural profiling of microbial communities. 16S rRNA genes are amplified from the community DNA and digested by a single restriction enzyme Msp I. All restriction fragments are end-labeled with a fluorescent nucleotide Cy5-dCTP via a one-step extension reaction and detected with an automated DNA sequencer. All 50 predicted restriction fragments between 100 and 600 bp were detected when twelve single 16S rRNA gene sequences were analyzed using f-DRT approach; 92% of these fragments were determined with accuracy of ±2 bp. In the defined model communities containing five components with different ratios, relative abundance of each component was correctly revealed by this method. The f-DRT analysis also showed structural shifts of intestinal microbiota in carcinogen-treated rats during the formation of precancerous lesions in the colon, as sensitive as multiple digestion-based T-RFLP analysis. This study provides a labor and cost-saving new method for monitoring structural shifts of microbial communities.
DNA 限制片段多态性技术，如扩增核糖体 DNA 限制分析（ARDRA）和末端限制片段长度多态性（T-RFLP），已广泛用于研究微生物群落结构。然而，这些方法由于分辨率或灵敏度低而受到限制。在本研究中，开发了一种荧光核糖体 DNA 限制分型（f-DRT）方法，用于微生物群落的结构分析。从群落 DNA 中扩增 16S rRNA 基因，并通过单一切割酶 Msp I 进行消化。所有限制片段均通过一步延伸反应用荧光核苷酸 Cy5-dCTP 末端标记，并通过自动 DNA 测序仪进行检测。当使用 f-DRT 方法分析 12 个单 16S rRNA 基因序列时，检测到了 100 至 600 bp 之间的所有 50 个预测限制片段；其中 92%的片段的精度为±2 bp。在含有不同比例的五个成分的定义模型群落中，该方法正确揭示了每个成分的相对丰度。f-DRT 分析还显示，在结肠癌癌前病变形成过程中，致癌物处理大鼠的肠道微生物群结构发生了变化，其敏感性与基于多次消化的 T-RFLP 分析相当。本研究为监测微生物群落结构变化提供了一种节省劳动力和成本的新方法。
