Development of oligonucleotide probes for rapid identification of diarrheagenic bacteria

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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.