Detection of point mutation in bla T genes of enterobacteriaceae by biotinylated oligonucleotide probes using microwell hybridization and enzymofluorometric method

Point mutation in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaden their substrate spectrum towards all β-lactams except cephamicins and imipenem. We describe here hybridization techniques for the detection of point mutations by non-radioactive oligonucleotide probes with plasmid DNA carrying bla T genes immobilized in polystyrene microwells. After hybridization in discriminating conditions with corresponding biotinylated oligonucleotide probes, the hybrids were detected by using a streptavidin-alkaline phosphatase conjugate and a fluorogenic substrate, 4-methylumbelliferyl-phosphate. The adsorption of DNA to microwells used in the present work was found to be independent of Mg+ and Na2+ concentrations. By this method, less than 3 fmols of target DNA were sufficient for the detection of point mutation.