利用串联重复序列研究炭疽芽胞杆菌的基因分型

目的 应用基因组中多位点串联重复序列遗传标记对不同地区88株炭疽芽胞杆菌进行基因分型。方法炭疽芽胞杆菌染色体DNA基因组中存在着串联重复序列。在串联重复序列两侧设计引物，PCR扩增，琼脂糖和聚丙烯酰胺凝胶电泳，凝胶影像分析软件对PCR扩增产物碱基含量进行测算，并与测序结果进行比较，计算出串联重复拷贝数，对拷贝数进行聚类分析。结果 （1）聚类分析发现，88株菌株可分为三大群，45个基因型，基因型与生态环境存在一定的关系。就某一地区炭疽暴发而言，其可变数目串联重复序列遗传标记具有相似性。（2）研究发现，A16R疫苗株作为中国的疫苗株具有代表性。结论 炭疽芽胞杆菌基因组中的串联重复序列具有遗传稳定性和特异性，可作为炭疽芽胞杆菌基因分型的指标，在炭疽暴发和生物恐怖事件中的病原体溯源上具有重要的意义。

Use of variable-number tandem repeats to examine genetic diversity of Bacillus anthracis

Objective To study the genotyping of Bacillus anthracis based on multiple-locus variable-number tandem repeats(VNTR) in the B . anthracis genome. Methods We selected 13 VNTR loci (which cited from published articles) to study 88 strains of B. anthracis isolated from China. The methods used were: (1)Selecting the primers which were at both ends of the tandem repeat locus; (2) Amplifying the sequence of the locus by PCR; (3) Detecting the PCR products by agarose gel and polyacrylamide electrophoresis; (4 ) Analyzing the PCR products and computing the molecular weight by analysis software of gel images; (5) Double-checking with sequencing results; (6) Reckoning the repeat numbers and study the VNTRs loci characters. Results (1) We used multiple-locus variable-number tandem repeat analysis (MLVA) to characterize 88 B. anthracis isolates from diverse geographic locations which were divided into 45 MLVA genotypes and 3 groups through cluster analysis. The genotypes was relative to restricted geographical region. It seemed clear that the multiple isolates from the same anthrax outbreak frequently having identical genotypes. (2)Results from VNTR analysis showed that A16R vaccine strain isolated from China was having the nature of representativeness in the country. Conclusion Analysis showed that the VNTR patterns was an appropriate study method for B. anthracis genetic diversity from different geographical areas and different time. Isolates from the same anthrax outbreak had identical VNTR genotyping.