炭疽芽孢杆菌MLVA分子分型研究

发布时间：2018-03-23 20:20

本文选题：炭疽芽孢杆菌　切入点：多位点串联重复序列　出处：《南昌大学》2011年硕士论文

【摘要】：炭疽病是由炭疽芽孢杆菌(Bacillus anthracis)引起的人兽共患传染病,曾多次被恐怖分子用来发动生物恐怖袭击。随着国际上生物战剂的研究发展和恐怖组织的活动频繁,炭疽越来越对人畜构成了威胁,因此建立快速、经济的分子分型方法,对预防和控制炭疽芽孢杆菌的大面积爆发和流行来说显得非常必要。本研究对我国的炭疽芽孢杆菌进行了可变数目串联重复序列分析(Multiple Locus Variable- Number Tandem Repeats Analysis, MLVA)分析,初步探讨了可变数目串联重复(Variable Number Tandem Repeat, VNTR)位点在炭疽芽孢杆菌中的多态性,以及中国地区的主要流行菌株之间的遗传关系。本文使用经典的苯酚氯仿方法,对全国各炭疽自然疫源地分离到的198株炭疽芽孢杆菌的基因组DNA进行了提取。利用Tandem Repeats Finder软件进行生物信息学分析后,最终选取了18个VNTR多态性位点。针对不同的VNTR多态性位点两端的基因组序列,设计了特异性荧光标记引物,PCR扩增后通过毛细管电泳检测。通过Genemarker软件确定PCR扩增片段长度,计算每个菌株不同串联重复位点拷贝数。采用Bionumerics软件分析,选用非加权组平均法(UPGMA)对炭疽芽孢杆菌进行聚类分析,研究炭疽芽孢杆菌菌株之间的遗传差异。结果表明：使用18个串联重复位点进行UPGMA聚类分型,可鉴定出96个基因型。按遗传学距离归纳为2个集群A和B,共分为4个组Al,A2,B 1,B2。A1组可划分为两个亚组A1.a,Ai.b,B1组可划分为两个亚组B1.a，，B1.b。B2组也可划分为两个亚组B2.a,B2.b。炭疽芽孢杆菌分布主要集中在A1.b亚群以及B1组和B2组中。A1.b亚群主要分布的基因型为4、18和31,Bl组主要分布的基因型为57、59、68、77和78,B2组主要分布的基因型为84和92。VNTR多态性位点多态性信息含量分析结果显示,Bams1、Bams34、A031、Bams3、VrrC1、Bams30、VrrC2、Bams31八个VNTR多态性位点具有较高的多态性,确定了一个新的VNTR多态性位点A031。选用这八个多态信息含量最高位点进行组合后对炭疽芽孢杆菌进行UPGMA聚类分析,可鉴定出66个基因型。本论文的初步研究结果将对推动我国炭疽芽孢杆菌的分子分型,保障我国在生物防控领域的监控水平,起到积极的推动作用。
[Abstract]:Anthrax is a zoonotic infectious disease caused by Bacillus anthracis. it has been used by terrorists to launch bioterrorist attacks many times. Anthrax is more and more threatening to human and animal, so it is necessary to establish a rapid and economical molecular typing method to prevent and control the outbreak and prevalence of Bacillus anthracis in a large area. In this study, the variable Locus variant-Number Tandem Repeats analysis (MLVA) analysis of Bacillus anthracis was carried out, and the polymorphism of variable Number Tandem repeat (VNTR) locus in Bacillus anthracis was preliminarily investigated. And the genetic relationship among the main endemic strains in China. The genomic DNA of 198 strains of Bacillus anthracis isolated from natural foci of anthrax in China were extracted by the classical phenol chloroform method. The bioinformatics analysis was carried out by using Tandem Repeats Finder software. Finally, 18 polymorphic VNTR loci were selected. Specific fluorescent marker primers were designed to amplify the genomic sequences at both ends of the VNTR polymorphism sites and detected by capillary electrophoresis. The length of the amplified PCR fragments was determined by Genemarker software. The copy number of different tandem repeat sites of each strain was calculated. The cluster analysis of Bacillus anthracis was carried out by using Bionumerics software and unweighted group averaging method to study the genetic difference among Bacillus anthracis strains. The results showed that 18 tandem repeats were used for UPGMA cluster typing. According to genetic distance, 96 genotypes were identified and divided into 4 groups: group A and B, divided into four groups: group A, A, A, A, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B, B. The main genotypes of A1.b subgroup and group B1 and B2 were 4N18 and 31Bl respectively. The main genotypes of A1.b subgroup were 57N59A9A6877 and 78FB 2 groups. The genotype distribution was 84 and the polymorphism information of 92.VNTR polymorphism was also found in group B 1 and group B 2. The results of content analysis showed that the eight VNTR polymorphism loci of Bams1, Bams34, A031, Bams3, VrrC1, Bams30, VrrC2, BamS31, were highly polymorphic. A new polymorphic VNTR locus A031 was identified. The UPGMA cluster analysis of Bacillus anthracis showed that 66 genotypes could be identified by the combination of the eight loci with the highest polymorphic information content. The preliminary results of this paper will play a positive role in promoting the molecular typing of Bacillus anthracis and ensuring the monitoring level in the field of biological prevention and control in China.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378;S852.616

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