Development of oligonucleotide probes for rapid identification of diarrheagenic bacteria

近年来，利用杂交技术检测致病菌毒力因子基因的存在与否已成为一种快速鉴定病原菌的新方法。然而，由于技术问题，这些方法尚未在临床实验室中普遍使用。本研究以解决这些问题和开发具有实用价值的探针为最终目的，尝试制备非同位素标记的合成寡核苷酸探针，用于鉴定大肠杆菌的热稳定肠毒素。主要研究结果如下：1.用代表大肠杆菌耐热肠毒素编码基因区域的寡核苷酸探针进行杂交试验。大肠杆菌中发现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E.大肠杆菌菌株产生具有生物活性的耐热肠毒素.我们开发了一种通过交联剂和间隔物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低至几十阿托摩尔的DNA。我们证明了产热稳定肠毒素的E。用人工合成的碱性磷酸酶标记的寡核苷酸探针进行DNA菌落杂交，可用于大肠杆菌的鉴定。

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.