Pulsed field gel electrophoresis protocol for typing of uropathogenic Escherichia Coli

Abstract

Introduction:Pulsed Field Gel Electrophoresis (PFGE) is among the best typing methods for epidemiologic studies in different areas. The high reproducibility and distinction of this method render it applicable to all human pathogens. Nevertheless, it is highly sensitive and the slightest variation in its associated factors will compromise the results. Therefore, in this study we have tried to improve the standardization of this method and circumvent its current technical challenges in order to achieve more accurate results for comparing different strains in epidemiologic studies.Material and Methods:In order to determine the genetic pattern of Escherichia coli strains using PFGE, we investigated different parameters such as DNA concentration in sample and its relationship with concentration and duration of enzyme use.Results:After different examinations, we found the optimal optical density of bacterial suspension at wavelength of 610 nm to be 0.6-0.7 nm, the XbaI enzyme concentration to be 150 units, and the optimal time for proteinase K effect to be 7 hours.Conclusion:It appears that the variables addressed in the present study constitute the major variables for optimizing PFGE. Thus, observing the findings of this study will help researchers to optimize their use of this molecular method in epidemiologic studies and comparison of different strains.

Keywords

• Escherichia coli
• Electrophoresis
• Gel
• Pulsed Field