Increased Reliability in Detection of Enterotoxigenic Escherichia coli by DNA Colony Hybridization ¹

Previously a DNA hybridization assay was designed to detect the presence of and to enumerate enterotoxigenic foodborne Escherichia coli . The determinative step in the method involves autoradiographic analysis of the DNA from foodborne isolates after hybridization with a ³²P-labeled probe specific for an enterotoxin gene. Dark spots appearing on the X-ray film after exposure indicate which colonies carry genes encoding the pathogenic determinant. A problem with this assay is the tendency of some colonies to detach from the nitrocellulose filters during hybridization or washing to remove the unbound probe DNA; this results in a false-negative interpretation in up to 60% of the samples processed at 80°C. By lowering the temperature to 70°C and increasing the incubation time to 3 h during in vacuo baking of filters, detachment (flotation) of colonies is reduced to about 37%. At 65°C only 2% of the colonies came off the filter after in vacuo baking of filters for 24 h. Another problem has been the inadequacy of exposure of X-ray film at -20°C when a -70°C freezer is not available. This problem can be alleviated by exposing the X-ray film in cassette holders "sandwiched" between slabs of dry ice (CO₂ ice has a temperature of -78.5°C). These modifications improve the reliability and accuracy of this DNA colony hybridization method.