一种五联重复序列在分枝杆菌简单序列重复区间分型中的应用研究

The study of inter.simple sequences repeat genotyping based on one pentanucleotide repeat sequencesin mycobacteria

目的分析微卫星5联简单重复序列在分枝杆菌基因组中的分布情况，并评价其简单序列重复区间（ISSR）分型能力，为分枝杆菌的基因分型及流行病学研究提供新的研究工具。方法实验菌株为17株分枝杆菌菌株和41株MTB临床分离株，17株分枝杆菌菌株包括分枝杆菌标准株15株、MTB临床菌株1株和MTB无毒株（H37Ra）1株。利用MICdb2．0软件分析微卫星数据库收录的分枝杆菌基因组中5联简单重复序列（CAGCG）n的分布特征，并以该序列为基础设计ISSR引物（5’-CAGCGCAGCGCAGCG-3’），用于分枝杆菌种间和种内菌株的基因分型分析。结果生物信息学分析显示（CAGCG）n在数据库收录的大部分分枝杆菌基因组中均有较高含量，其中在H37Rv、CDCl551、牛分枝杆菌和鸟分枝杆菌基因组中出现的次数分别为40、39、39和33次，且主要分布在编码序列区（分别为39、36、38和30次）。ISSR引物对分枝杆菌菌种的基因分型分析显示，15种常见菌种聚为2大簇，第1簇含有2个亚型，第2簇含有4个亚型，种间表现出高度的遗传多样性；ISSR引物对MTB临床菌株的分析结果显示，41株菌株聚为2簇，各簇分别有2个亚型，提示该引物对种内菌株也表现出良好的分辨力。结论以（CAGCG）n建立的ISSR分型体系，可用于分枝杆菌种间和种内菌株的遗传多样性分析。

Objective To establish inter-simple sequences repeat （ISSR） molecular makers based on （CAGCG） n repeat sequence in mycobacteria. Methods The distribution of pentanucleotide repeat sequence （CAGCG）n in mycobacterial genomes was analyzed by MICdb 2. 0 software in the microsatellite database. ISSR primer MISP6 based on （CAGCG）n sequences was designed and tested in mycobacterial strains, which included 17 mycobacterial strains and 41 Mycobacterium tuberculosis clinical strains. Results The abundances of pentanucleotide repeat sequences （CAGCG） n were high in most of the mycobacterial genomes and they were mainly located in the coding regions. The results of ISSR analysis in mycobacteria showed that 15 reference strains from mycobacteria were clustered into 2 major clusters. The first cluster contained 2 subtypes and the second cluster contained 4 subtypes. Forty-one clinical strains from Mycobacterium tuberculosis were divided into 2 major clusters by the analysis of MISP6 primer, and each cluster had 2 subtypes. Conclusion ISSR primer MISP6 based on （CAGCG） n sequences can be used as a genetic marker to geuotype mycobacterial strains.