High-resolution differentiation of cyanobacteria by using rRNA-internal transcribed spacer denaturing gradient gel electrophoresis
High-resolution differentiation of cyanobacteria by using rRNA-internal transcribed spacer denaturing gradient gel electrophoresis
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DOI：
10.1128/aem.69.11.6634-6643.2003
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发表时间：
2003-11-01
影响因子：
4.4
通讯作者：
Zwart, G
中科院分区：
文献类型：
--
作者：
Janse, I;Meima, M;Zwart, G
对于蓝藻的许多生态学研究，可以区分密切相关的物种或菌株是必不可少的。由于这往往是不可能的，通过使用形态特征，蓝藻经常使用DNA为基础的方法进行研究。变性梯度凝胶电泳（DGGE）是分析微生物种群多样性和动态以及检查培养菌株纯度和从属关系的一种有效方法。利用DGGE技术分析了蓝藻16 S和23 S rRNA基因间的内转录间隔区（rRNA-ITS），实现了蓝藻的高分辨率鉴别。设计了一条针对蓝藻的正向引物，定位于16 SrRNA基因的3'端。该引物和靶向rRNA-ITS或23 S rRNA基因的三种不同的反向引物的组合产生了来自所有16种蓝藻属的培养物的不同大小的PCR产物，所述蓝藻属被测试，但不是来自其他细菌。从最短的一节的rRNA-ITS产生的DGGE配置文件包括一个带的所有，但一个蓝藻属，和那些产生的较长的延伸的rRNA-ITS产生的DGGE配置文件包含一个到四个带。DGGE技术检测微囊藻属内和种内变异的适用性进行了测试。利用rRNA-ITS DGGE技术可以对多种菌株进行区分，其分辨率明显高于以往的方法。通过使用淡水湖泊样品证明了所开发的DGGE检测用于分析现场样品中的蓝藻的适用性。与使用每个开发的引物集的优点和缺点进行了讨论。
For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3' end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.