分支杆菌临床分离株的16S-23S rRNA基因转录间隔区的序列分析

目的　建立鉴定分支杆菌分离株的 16S - 2 3SrDNA转录间隔区 (internaltranscribedspacer ,ITS)序列分析的方法 ,用该方法对临床分离株进行鉴定。方法　对从重庆某医院分离的 2 9株分支杆菌菌株分别进行了PCR扩增 ,得到它们的16S - 2 3SrDNAITS片段 ,并测定所得到片段的DNA序列。用DNA分析软件将所获得的序列与GenBank的分支杆菌序列相比较 ,计算种间相似性 ,并用序列构建分支杆菌菌株的系统发育树 ,对分离株进行鉴定。结果　在ITS序列分析中 ,有 10株的序列分别与结核分支杆菌复合群 (Mycobacteriumtuberculosiscomplex ,MTC)、戈登分支杆菌 ,脓肿分支杆菌的序列完全一致。依据完全一致的序列可以准确鉴定这些临床分离株为相应的种 ;4株 16SrDNA序列分析不能准确鉴定的分离株中其中 3株(M 4、M 5、M 12 )鉴定为戈登分支杆菌 ,1株 (M 2 3)鉴定为脓肿分支杆菌。部分分离株的的ITS序列在GenBank序列检索中无完全一致的匹配序列 ,这些不同的序列之间的相似程度为 2 7.1%～ 99.2 %。用临床分离株的ITS序列与其最相似的已知分支杆菌的ITS序列构建的系统发育树 ,菌株分群与 16SrDNA序列构建的系统发育树的分群基本一致。结论　分支杆菌的16S - 2 3SrDNAITS序列比 16SrDNA序列有更大的多样性 ,该序列分析可作为

Sequence analysis of the internal gene transcribed spacers of 16S-23S rRNA in clinical isolates of mycobacteria

To establish a method of sequence analysis to identify the internal gene transcribed spacers (ITS) of 16S rRNA in the clinical isolates of mycobacteria.The ITS fragment of 19 clinical isolates from sputa of patients with mycobacterial infections in a hospital in Chongqing were amplified by PCR, and the amplified fragments were directly sequenced. The sequences of the isolates were compared with those in GenBank with soft wares for DNA analysis. Experimental results showed that the levels of the ITS sequences in different isolates were quite different, and ranged from 27.1% to 99.2%. The ITS sequences of 10 strains were identical to those of Mycobacterium tuberculosis complex (MTB), M.gordanae and M.abscessus, and the ITS sequences in some isolates were not similar to any sequence in GenBank. Four isolates unidentified in the 16S rRNA analysis were proved to be M.gordanae(M4,M5,M12) or M.abscessus (M23) by the 16S 23S rRNA ITS sequence analysis. It is concluded that the sequences of ITSs of mycobacteria are much more mutable than the 16S rRNA sequences, and the sequence analysis with 16S 23S rRNA ITS may be used as a supplement for the 16S rDNA analysis in the identification of mycobacteria.