Development of a fluorophore-ribosomal DNA restriction typing method for monitoring structural shifts of microbial communities
Development of a fluorophore-ribosomal DNA restriction typing method for monitoring structural shifts of microbial communities
复制标题
开发用于监测微生物群落结构变化的荧光团-核糖体 DNA 限制性分型方法
DOI：
10.1007/s00203-011-0679-8
复制
发表时间：
2011-05-01
影响因子：
2.8
通讯作者：
Zhao, Liping
中科院分区：
文献类型：
--
作者：
Wang, Tingting;Zhang, Xiaojun;Zhao, Liping
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.
