Pulsed‐Field Gel Electrophoresis: Laboratory and Epidemiologic Considerations for Interpretation of Data

Abstract

This chapter reviews pulsed-field gel electrophoresis (PFGE) as an epidemiological tool, considering (i) factors that influence the electrophoretic process, (ii) methodological streamlining, (iii) the troubleshooting of common problems, (iv) quality assurance, (v) use of PFGE for continuous surveillance, and (vi) issues of data interpretation. To be suitable for reliable PFGE analysis, intact chromosomal DNA must be isolated in a protected environment free from mechanical, chemical, and enzymatic degradation to yield a clear and reproducible macrorestriction fragment pattern. As PFGE analysis is applied to larger study populations, the need for computer-assisted analysis (CAA) of banding patterns becomes increasingly evident. At the laboratory level the quality assurance/ quality control (QA/QC) system consists of strict adherence to each of the PFGE standard operating procedures (SOPs) as described in the laboratory QA/QC manual. It is important to emphasize that the successful establishment of dynamic databases is dependent on strict adherence to well-defined QA and QC criteria. An important component of the protocol standardization and QA/QC program for PulseNet is the annual update meeting. Molecular typing, along with a variety of other microbiological assays is clearly moving toward sequence-based analysis. However, this approach is still being validated for a variety of applications including strain typing. Thus far, none of the new sequence-based typing methods are as broadly applicable as PFGE. Therefore, while this problem will undoubtedly be solved in the future, at present PFGE will clearly continue to provide meaningful epidemiological data on molecular typing in a variety of important settings for years to come.