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 coli for virulence genes, a Phosphor Imager was adopted for signal detection in Dot 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 going from an outcome measured by visualization on a semi-quantitative scale to a digitized readout on an interval scale. We analyzed technical and biological variability of the assay and the factors contributing to the variability. In spite of high variability both within and between membranes in the Phosphor Imager readings, we were able to define classification rules for gene presence that were remarkably consistent. Using the X-ray film signal detection procedure with Southern confirmation as a gold standard, we obtained a sensitivity and specificity of 87-99% for a rule requiring no retesting for all but one gene probe.
Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI 48109, USA.
L
L
Zhang
B W
BW
Gillespie
C F
CF
Marrs
B
B
Foxman
D001345:Autoradiography; D015139:Blotting, Southern; D015342:DNA Probes / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000472:pathogenicity*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015588:Observer Variation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014774:Virulence / Q000235:genetics
D015342:DNA Probes; D004269:DNA, Bacterial