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Development of oligonucleotide probes for rapid identification of diarrheagenic bacteria

开发用于快速鉴定致腹泻细菌的寡核苷酸探针

基本信息

批准号：
62870019
负责人：
NISHIBUCHI Mitsuaki
金额：
$ 2.43万
依托单位：
Kyoto University (1988)Osaka University (1987)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-62870019/
关键词：
Probe Enterotoxigenic Escherichia coli Heat-stable Enterotoxin ハイブリダイゼーション

项目摘要

Hybridization methods to detect presence or absence of the genes encoding virulence factors have been introduced recently for rapid identification of pathogenic bacteria. The methods, hoerver, have not been put to ordinary use in clinical laboratories due to technical problems. In this study, with the ultimate intention to solve the problems and develop probes of practical value, we attempted to make nonisotopically labeled synthetic oligonucleotide probes for identifying heat stable enterotixins of Escherichia coli. The results obtained are summarized as follows:1. Hybridization tests using oligonucleotide probes representing regions of the gene encoding the heat-stable enterotoxins of E. coli revealed a region of the gene which is associated with enterotoxic activity. An oligonucleotide probe for this region detected E. coli strais producing biologically active heatstable enterotoxins.2. We deceloped a method to label synthetic oligonucleotide probes with alkaline phosphatase via a crosslinker and spacer. The labeled oligonucleotide probes detected as low as several tens of attomoles of DNA.3. We demonstrated that the heat stable enterotoxin-producing E. coli strains could be identified by a simple and rapid DNA colony hybridization method with a synthetic oligonucleotide probe laveled with alkaline phosphatase.

最近引入了检测编码毒力因子的基因是否存在的杂交方法，以快速鉴定病原菌。然而，由于技术问题，这些方法尚未在临床实验室中得到普遍应用。本研究的最终目的是解决问题并开发具有实用价值的探针，我们尝试制备非同位素标记的合成寡核苷酸探针来鉴定大肠杆菌的热稳定性肠毒素。所得结果总结如下： 1．使用代表编码大肠杆菌热稳定性肠毒素的基因区域的寡核苷酸探针的杂交测试揭示了与肠毒性活性相关的基因区域。该区域的寡核苷酸探针检测到产生具有生物活性的耐热肠毒素的大肠杆菌菌株。2．我们开发了一种通过交联剂和间隔基用碱性磷酸酶标记合成寡核苷酸探针的方法。标记的寡核苷酸探针检测到的DNA低至几十阿托摩尔。3．我们证明，可以通过简单快速的 DNA 菌落杂交方法，使用涂有碱性磷酸酶的合成寡核苷酸探针来鉴定热稳定的产肠毒素大肠杆菌菌株。