Comparative genome analysis & enterohemorrhagic Escherichia coli O157 and its clinical application.

比较基因组分析

基本信息

批准号：
13470061
负责人：
HAYASHI Tetsuya
金额：
$ 8万
依托单位：
Miyazaki Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470061/
关键词：
enterohemorrhagic Escherichia coli O157 genome diversity bacteriophage PCR strain-discrimination system

项目摘要

The aims of the project were to reveal the genomic diversity of Escherichia coli O157, and to develop a new tool for molecular epidemiological studies of O157 infection based on the genomic diversity.We first selected eight O157 strains that exhibited diverse Xbal-digestion patterns from 1796 strains isolated in Japan in 1998. Next, based on the genome sequence of O157 Sakai, we prepared a set of PCR primers to amplify 560 DNA segments covering the whole genome of O157 (the average length of segments was about 10 Kb). By using long PCR and the primer set, we analyzed the whole genome structures of the eight O157 strains using the Sakai strain as a reference. By this analysis which we named Whole Genome PCR Scanning (WGPS), we could identify all the genomic regions with any structural polymorphism, indicating that WGPS is a powerful technique for the comparative analysis of closely related genomes. The data also indicate that an unexpectedly high degree of genomic diversity exists among O157 strains. In particular, prophage regions, including Stx-transducing phages, exhibited extensive diversities.By the WGPS analysis, about 30 genomic regions exhibiting small-size structural polymorphisms detectable by conventional PCR were also identified. Selecting 9 target regions among them, we constructed a multiplex PCR system. Since the stx1, stx2 and eae genes were also included as targets, the system was finally composed of 12 PCR reactions. In our preliminary examination, this system classified 51 strains into 21 types, suggesting that the system could be developed as a useful tool for the epidemiological studies, but some improvement is still required.In addition, we performed comparative and functional analyses of some O157 genes, and found that urease genes are specifically distributed to strains belonging to enterohemorrhagic E. coli, and that the toxB gene is involved in epithelial cell adherence.

该项目的目的是揭示大肠杆菌O157的基因组多样性，并开发一种基于基因组多样性的O157感染的分子流行病学研究的新工具，我们首先选择了八种O157菌株，这些菌株在1998年的日本中脱离了1796型菌株的多样性Xbal-digestion sequone 7 seploye o ewerne of necte o necte o necte o necte o e Next of of ocy pers of necte o e Next of。一组PCR引物扩增560个DNA片段，涵盖了O157的整个基因组（平均段的平均长度约为10 kb）。通过使用长PCR和底漆集，我们使用Sakai菌株作为参考分析了八个O157菌株的整个基因组结构。通过我们将整个基因组PCR扫描（WGP）命名的分析，我们可以鉴定出所有具有任何结构多态性的基因组区域，表明WGPS是对紧密相关基因组进行比较分析的强大技术。数据还表明，O157菌株之间存在意外的高度基因组多样性。特别是，在WGPS分析中，还发现了大约30个基因组区域，表现出可通过常规PCR检测到的小型结构多态性，包括大约30个基因组区域。在其中选择了9个目标区域，我们构建了一个多重PCR系统。由于STX1，STX2和EAE基因也被包括为靶标，因此该系统最终由12个PCR反应组成。 In our preliminary examination, this system classified 51 strains into 21 types, suggesting that the system could be developed as a useful tool for the epidemiological studies, but some improvement is still required.In addition, we performed comparative and functional analyses of some O157 genes, and found that urease genes are specifically distributed to strains belonging to enterohemorrhagic E. coli, and that the toxB gene is involved in epithelial cell坚持。