Kim B J, Lee S H, Lyu M A, Kim S J, Bai G H, Chae G T, Kim E C, Cha C Y, Kook Y H
Department of Microbiology and Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799, Korea.
J Clin Microbiol. 1999 Jun;37(6):1714-20. doi: 10.1128/JCM.37.6.1714-1720.1999.
For the differentiation and identification of mycobacterial species, the rpoB gene, encoding the beta subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were directly determined (306 bp) and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Comparative sequence analysis of rpoB DNAs provided the basis for species differentiation within the genus Mycobacterium. Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. Pathogenic Mycobacterium kansasii was easily differentiated from nonpathogenic M. gastri; this differentiation cannot be achieved by using 16S rRNA gene (rDNA) sequences. By being grouped into species-specific clusters with low-level sequence divergence among strains of the same species, all of the clinical isolates could be easily identified. These results suggest that comparative sequence analysis of amplified rpoB DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. Furthermore, in the case of M. tuberculosis, rifampin resistance can be simultaneously determined.
为了对分枝杆菌属菌种进行鉴别和鉴定，研究了编码RNA聚合酶β亚基的rpoB基因。通过PCR从44株分枝杆菌参考菌株和临床分离株（107株）中扩增出rpoB DNA（342 bp）。直接测定核苷酸序列（306 bp），并使用MegAlign软件包（DNASTAR）中的多重比对算法和MEGA程序进行比对。采用邻接法构建系统发育树。rpoB DNA的比较序列分析为分枝杆菌属内的菌种鉴别提供了依据。分枝杆菌的缓慢生长组和快速生长组被明显分开，并且每个分枝杆菌菌种在系统发育树中都被鉴别为一个独特的实体。致病性堪萨斯分枝杆菌很容易与非致病性胃分枝杆菌区分开来；使用16S rRNA基因（rDNA）序列无法实现这种区分。通过将临床分离株分组到同一菌种菌株间序列差异水平较低的种特异性簇中，可以很容易地对所有临床分离株进行鉴定。这些结果表明，扩增的rpoB DNA的比较序列分析可以与传统培养方法并行有效地用于鉴定分枝杆菌临床分离株，并作为16S rDNA基因分析的补充。此外，对于结核分枝杆菌，还可以同时测定利福平耐药性。
