16S～23S rDNA内转录间隔区序列分析及其在分支杆菌鉴定中的应用价值

目的：研究16S－23SrDNA内转录间隔区（internal transcribed spacr,ITS)序列在分支杆菌鉴定中的应用价值。方法：应用PCR－直接测序法对22例30株分支杆菌参考菌株和16株分支杆菌临床分离菌株16S－23SrDNA ITS测序。对所测定序列以及GenBank所报道的有关序列用Clustal程序（MegAlign Package[Windows Version4.01]；DNASTAR，Madson,Wis)处理分析，计算种间相似性，用PHYLIP Package构建分支杆菌菌种聚类分析树状谱。结果：除结核分支杆菌复合群（MTC）菌种（结核分支杆菌、牛分支杆菌、非洲分支杆菌和田鼠分支杆菌）16S－23SrDNA ITS序列完全一致外，其它分支杆菌菌种间核苷酸排列顺序及长度变异较大，种间相似性为30．4％－86．5％，聚类分析树状谱能将各分支杆菌菌种相分离，结果表明16S－23SrDNA ITS序列分析能将MTC与非结核分支杆菌（NTM）相鉴别，能将NTM鉴定至种的水平。结论：16S－23SrDNA ITS可做为分支杆菌鉴定的靶基因。

16S～23S rDNA internal transcribed spacer sequence analysis and its application in mycobacterial identification

OBJECTIVE: To study the applicability of the 16S approximately 23S rDNA internal transcribed spacer (ITS) sequences in mycobacterial identification. METHODS: The 16S approximately 23S rDNA ITS sequences of 22 mycobacterial species (30 reference strains and 16 clinical isolates) were determined by PCR-direct sequencing. The sequences determined by this study and those reported in Genbank were analysed by Clustal program (MegAlign Package [Windows Version 4.01]; DNASTAR, Madison, Wis). The percentage similarity of sequence pairs was calculated and a cluster analysis dendrogram was constructed by PHYLIP package. RESULTS: The 16S approximately 23S rDNA ITS sequences among mycobacterial species had highly polymorphism except that Mycobacterium tuberculosis complex (MTC) sequences were highly consistent. The similarity between sequence pairs was 30.4% to 86.5%. Mycobacterial species could be isolated by cluster analysis dendrogram. The results showed that sequence analysis of the 16S approximately 23S rDNA ITS can differentiate MTC from nontuberculous mycobacteria (NTM) and identify NTM at species level. CONCLUSION: 16S approximately 23S rDNA ITS sequences can be used as target genes in mycobacterial identification.