幽门螺杆菌中国菌株基因型分析

发布时间：2018-04-22 19:05

本文选题：幽门螺杆菌 + 多位点序列分型　；参考：《天津医科大学》2011年硕士论文

【摘要】：目的：研究幽门螺杆菌(H. pylori)中国分离株的基因分型特征。方法：对分离自中国7个不同地区(北京、黑龙江、烟台、浙江、湖北、西安、云南)、不同胃、十二指肠相关疾病(慢性胃炎、消化性溃疡、胃癌、功能性消化不良)患者的119株H.pylori的7个看家基因(atpA, efp, mutY,ppa, trpC, urel和yphC)和毒力相关基因vacA,应用多位点序列分型(multilocus sequence typing, MLST)的方法获得菌株的等位基因谱(allelic profile)和序列型(sequnce type,ST),与HP MLST数据库中世界范围内的菌株信息进行比较。应用软件BURST (Based Upon Related Sequence types)运算法则将菌株归为谱系(clonal complex),应用软件START2 (Sequence Type Analysis and Recombinational Tests, version 2)计算相关系数(Index of Association, IA),应用软件MEGA4.0(Molecular Evolutionary Genetics Analysis, version 4.0)构建进化树进行分子进化遗传分析。对vacA等位基因(sla, slb, s2, mla, mlb, m2, il和i2)进行特异引物聚合酶链式反应(polymerase chain reaction, PCR),根据扩增产物片段大小确定vacA基因型,对基因型分布应用SPSS13.0软件数据包进行统计学分析、并应用层级聚类分析软件Mev_4_0 (Multiple Experiment Viewer, TIGR)进行聚类分析。结果：MLST共确定了107个序列型,都是HP MLST数据库中未曾报道过的。119株H.pylori菌株被分为3个谱系,其他均为独特型。START2重组分析显示,107个STs的IA为2.1398。MEGA4.0软件将119株中国菌株分布在3个祖先群。111株分布在hpEastAsia,6株分布在hpEurope,2株分布在hpAsia2。119株H.pylori vacA基因以sla,m2和i1型为主,分别为97.5%(116/119),68.9%(82/119)和91.6%(109/119)。26.1%(31／119)为mlb。slb和mla未检出。vacA组合基因型以sla/m2/il为主(62.2%,74/119),sla/mlb/il次之(25.2%,30/119)。不同地域、不同疾病来源菌株sla分布差异无统计学意义(P0.05)。而m区基因多态性在疾病类型及分离地区间差异均有统计学意义(P0.01)。不同疾病来源菌株间i区基因分型差异无统计学意义(P0.05),但不同分离地区菌株间差异有统计学意义(P0.01)。119株H.pylori vacA基因序列聚类为三个不同组群。一株独在一个组群,8株在另一个组群,其余的菌株聚为一群。结论：本研究中H.pylori中国分离株绝大多数起源于hpEastAsia,很少部分属于hpAsia2(?)和hpEurope,尚未发现有起源于非洲的菌株。H.pylori中国分离株vacA基因型以sla m2 il组合型为主,sla分型结果与菌株分离地域及患者疾病类型无关。不同地域、不同疾病来源菌株m区基因分型不同。不同地域菌株vacA基因i区分型不同,但i区基因分型与菌株的疾病来源没有显著相关性。中国H.pylori菌株有一定的地域相关性,但疾病相关性不明显。
[Abstract]:Objective: to study the genotyping characteristics of Helicobacter pylori H. pylori isolates from China. Methods: isolated from 7 different regions of China (Beijing, Heilongjiang, Yantai, Zhejiang, Hubei, Xi'an, Yunnan, different stomach and duodenal diseases (chronic gastritis, peptic ulcer, gastric cancer). Seven housekeeping genes of 119 H.pylori strains from patients with functional dyspepsia (H.pylori) were identified by multilocus sequence typing (MLSTs) and virulence related genes (Vaca) and virulence related genes (Vaca). The allelic and serotype sequnce type STs were obtained by multilocus sequence typing (MLSTs). The results were compared with the worldwide strain information in HP MLST database. The phylogenetic analysis was carried out by using the algorithm of BURST based Upon Related Sequence types, and START2 sequence Type Analysis and Recombinational tests, version 2, MEGA4.0(Molecular Evolutionary Genetics Analysis, version 4.0) was used to calculate the correlation coefficient of association, IAA, MEGA4.0(Molecular Evolutionary Genetics Analysis, version 4.0) to construct the phylogenetic analysis. The phylogenetic analysis was carried out by using the software START2 sequence Type Analysis and Recombinational tests (version 2) to calculate the correlation index of association, IAA, MEGA4.0(Molecular Evolutionary Genetics analysis, version 4.0). Specific primer polymerase chain reaction (PCR) was performed on the alleles of vacA, such as SLAA, SLB, S2, mla, MLB, m2il and i2). The genotypes of vacA were determined according to the fragment size of the amplified products, and the distribution of vacA genotypes was analyzed statistically by SPSS13.0 software. The hierarchical cluster analysis software Mev_4_0 multiple Experiment Viewer (TIGR) was applied to cluster analysis. Results A total of 107 sequence types were identified, all of which were not reported in HP MLST database. 119 H.pylori strains were divided into 3 lineages. The recombination analysis of Idiotype. START2 showed that 107 STs IA software distributed 119 Chinese strains in 3 progenitor groups. 111 strains distributed in hpEastAsiaS6 strains distributed in hpAsia2.119 strain H.pylori vacA gene mainly distributed in hpAsia2.119 strain SLAM 2 and I 1 type. 97.5% 11.6 / 119 / 68.9 / 82 / 119 and 91.6 / 109 / 119 / 26.1 / 31 / 119 respectively. For mlb.slb and mla, the main genotype of Vaca was 62.2% / 119slar / 119slar / ml / ml / 119g, and 25.2nb / 119b / r 25.2nb / 119a, respectively, for mlb.slb and mla with no Vaca genotype 62.2% / 119slar / ml / r / il. There was no significant difference in the distribution of sla between different regions and different disease sources (P 0.05). But the gene polymorphism in m region was significantly different in disease type and isolation region (P 0.01). There was no significant difference in I region genotyping among strains from different disease sources, but there were significant differences in H.pylori vacA gene sequences among different isolates from different regions. There were three different groups of H.pylori vacA gene sequences. One strain was isolated in one group, 8 strains in another group, and the other strains were clustered into a group. Conclusion: most of the Chinese isolates of H.pylori in this study originated from hpEastAsia, and few of them belong to hpAsia2. No vacA genotypes of H.pylori strains originated in Africa were found in China, and sla m2il combinations were the dominant genotypes. The results were not related to the region of isolates and the disease types of patients. The genotyping of m region was different in different regions and different disease sources. The genotype of vacA gene I was different in different regions, but there was no significant correlation between the genotype of I region and the source of disease. H.pylori strains in China have a certain geographical correlation, but the disease correlation is not obvious.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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