Baele Margo, Stakenborg Tim, Lopes dos Santos Santiago Guido, Haesebrouck Freddy, Vaneechoutte Mario
Laboratory for Veterinary Microbiology, Department of Pathology, Bacteriology and Poultry Diseases, Ghent University, 9000 Gent, Belgium.
Cold Spring Harb Protoc. 2009 Apr;2009(4):pdb.prot5196. doi: 10.1101/pdb.prot5196.
Transfer RNA intergenic spacer length polymorphism analysis (tDNA-PCR) is a simple and reproducible polymerase chain reaction (PCR) technique for identification of bacteria at the species or even subspecies level. The primers used in the PCR are based on conserved sequences located at the edges of the tRNA genes. Because the selected consensus primers are directed outwardly, the intergenic spacers are amplified rather than the genes themselves. With each PCR, several amplicons of different lengths are obtained, because several intergenic spacers are present in each bacterial genome. The patterns thus obtained are identical within species, but differ between distinct species, and as a result, can be used for identification of bacterial species. The amplicons are separated using high-resolution (1 bp) electrophoresis (e.g., fluorescent capillary electrophoresis) and immediately digitized as tables composed of numerical lengths (expressed in base pairs) and peak intensities. For identification, the resulting peak pattern can be compared with a large database of patterns of well-identified bacterial strains, using an in-house-developed software package that is available online. New patterns (linked to the correct species name, which can be obtained, e.g., after 16S rRNA gene sequence determination) can be added to expand the database further. This protocol describes tDNA-PCR, followed by automated fluorescent capillary electrophoresis to identify bacterial species.
转运RNA基因间间隔区长度多态性分析（tDNA-PCR）是一种简单且可重复的聚合酶链反应（PCR）技术，用于在物种甚至亚种水平上鉴定细菌。PCR中使用的引物基于位于tRNA基因边缘的保守序列。由于所选的共有引物向外定向，因此扩增的是基因间间隔区而非基因本身。每次PCR都会获得几个不同长度的扩增子，因为每个细菌基因组中都存在几个基因间间隔区。这样获得的模式在物种内是相同的，但在不同物种之间有所不同，因此可用于鉴定细菌物种。使用高分辨率（1 bp）电泳（例如荧光毛细管电泳）分离扩增子，并立即将其数字化为由数字长度（以碱基对表示）和峰强度组成的表格。为了进行鉴定，可以使用在线提供的内部开发软件包，将所得的峰模式与大量已明确鉴定的细菌菌株模式数据库进行比较。可以添加新的模式（与正确的物种名称相关联，例如在16S rRNA基因序列测定后可以获得）以进一步扩展数据库。本方案描述了tDNA-PCR，随后进行自动荧光毛细管电泳以鉴定细菌物种。
