本文選題：炭疽芽孢桿菌　切入點(diǎn)：多位點(diǎn)串聯(lián)重復(fù)序列　出處：《南昌大學(xué)》2011年碩士論文

【摘要】：炭疽病是由炭疽芽孢桿菌(Bacillus anthracis)引起的人獸共患傳染病,曾多次被恐怖分子用來發(fā)動(dòng)生物恐怖襲擊。隨著國際上生物戰(zhàn)劑的研究發(fā)展和恐怖組織的活動(dòng)頻繁,炭疽越來越對(duì)人畜構(gòu)成了威脅,因此建立快速、經(jīng)濟(jì)的分子分型方法,對(duì)預(yù)防和控制炭疽芽孢桿菌的大面積爆發(fā)和流行來說顯得非常必要。 本研究對(duì)我國的炭疽芽孢桿菌進(jìn)行了可變數(shù)目串聯(lián)重復(fù)序列分析(Multiple Locus Variable- Number Tandem Repeats Analysis, MLVA)分析,初步探討了可變數(shù)目串聯(lián)重復(fù)(Variable Number Tandem Repeat, VNTR)位點(diǎn)在炭疽芽孢桿菌中的多態(tài)性,以及中國地區(qū)的主要流行菌株之間的遺傳關(guān)系。 本文使用經(jīng)典的苯酚氯仿方法,對(duì)全國各炭疽自然疫源地分離到的198株炭疽芽孢桿菌的基因組DNA進(jìn)行了提取。利用Tandem Repeats Finder軟件進(jìn)行生物信息學(xué)分析后,最終選取了18個(gè)VNTR多態(tài)性位點(diǎn)。針對(duì)不同的VNTR多態(tài)性位點(diǎn)兩端的基因組序列,設(shè)計(jì)了特異性熒光標(biāo)記引物,PCR擴(kuò)增后通過毛細(xì)管電泳檢測(cè)。通過Genemarker軟件確定PCR擴(kuò)增片段長度,計(jì)算每個(gè)菌株不同串聯(lián)重復(fù)位點(diǎn)拷貝數(shù)。采用Bionumerics軟件分析,選用非加權(quán)組平均法(UPGMA)對(duì)炭疽芽孢桿菌進(jìn)行聚類分析,研究炭疽芽孢桿菌菌株之間的遺傳差異。 結(jié)果表明：使用18個(gè)串聯(lián)重復(fù)位點(diǎn)進(jìn)行UPGMA聚類分型,可鑒定出96個(gè)基因型。按遺傳學(xué)距離歸納為2個(gè)集群A和B,共分為4個(gè)組Al,A2,B 1,B2。A1組可劃分為兩個(gè)亞組A1.a,Ai.b,B1組可劃分為兩個(gè)亞組B1.a，，B1.b。B2組也可劃分為兩個(gè)亞組B2.a,B2.b。炭疽芽孢桿菌分布主要集中在A1.b亞群以及B1組和B2組中。A1.b亞群主要分布的基因型為4、18和31,Bl組主要分布的基因型為57、59、68、77和78,B2組主要分布的基因型為84和92。VNTR多態(tài)性位點(diǎn)多態(tài)性信息含量分析結(jié)果顯示,Bams1、Bams34、A031、Bams3、VrrC1、Bams30、VrrC2、Bams31八個(gè)VNTR多態(tài)性位點(diǎn)具有較高的多態(tài)性,確定了一個(gè)新的VNTR多態(tài)性位點(diǎn)A031。選用這八個(gè)多態(tài)信息含量最高位點(diǎn)進(jìn)行組合后對(duì)炭疽芽孢桿菌進(jìn)行UPGMA聚類分析,可鑒定出66個(gè)基因型。 本論文的初步研究結(jié)果將對(duì)推動(dòng)我國炭疽芽孢桿菌的分子分型,保障我國在生物防控領(lǐng)域的監(jiān)控水平,起到積極的推動(dòng)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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R378;S852.616

【共引文獻(xiàn)】

相關(guān)期刊論文 前2條

1 左庭婷;端青;;MLVA和SNP分析在炭疽芽孢桿菌基因分型中的應(yīng)用[J];軍事醫(yī)學(xué)科學(xué)院院刊;2010年03期

2 吳松羽;劉先凱;宋麗;魏華;王恒j;;炭疽芽孢桿菌分子分型研究進(jìn)展[J];生物技術(shù)通訊;2011年05期

本文編號(hào)：1655035