Optimization of a fluorescent-based phosphor imaging dot blot DNA hybridization assay to assess E-coli virulence gene profiles

To increase the efficiency and consistency in screening Escherichia coil fo r virulence genes, a Phosphor Imager was adopted for signal detection in Do t Blot DNA hybridization replacing X-ray film read by eye. We assessed not only the reliability of the instrument-based procedure, but the impact of g oing from an outcome measured by visualization on a semi-quantitative scale to a digitized readout on an interval scale. We analyzed technical and bio logical variability of the assay and the factors contributing to the variab ility. In spite of high variability both within and between membranes in th e Phosphor Imager readings, we were able to define classification rules for gene presence that were remarkably consistent. Using the X-ray film singal detection procedure with Southern confirmation as a gold standard, we obta ined a sensitivity and specificity of 87-99% for a rule requiring no retest ing for all but one gene probe. (C) 2001 Published by Elsevier Science B.V.