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Chapter 10 : Pulsed-Field Gel Electrophoresis: Laboratory and Epidemiologic Considerations for Interpretation of Data
Category: Clinical Microbiology; Bacterial Pathogenesis
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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.
SmaI-digested chromosomal DNA from six isolates of MRSA analyzed by PFGE at 6 V/cm, 14°C, 120° included angle, with switching from 1 to 34 s for 22 h (A) or 1 to 10 s for 12 h (B). Lane B6 is the 1-kb DNA ladder (Invitrogen Life Technologies, Rockville, MD).
Illustration of common problems in PFGE analysis including incomplete restriction-endonuclease digestion (A, lanes 2 to 4) and electrophoresis-induced degradation of banding patterns (B, lanes 2 to 5) corrected by inclusion of 75 µM thiourea in the PFGE running buffer (C).
PFGE gel showing the PulseNet global reference standard, XbaI-restricted Salmonella enterica serotype Braenderup H9812, in lanes 1, 5, and 10. The placement of the reference standard is critical for proper normalization and analysis of patterns generated in different laboratories.
Diagrammatic representation of different chromosomal changes which may or may not involve a rare restriction site (A) and the influence these genetic events would have on PFGE restriction-fragment analysis (B).
PFGE analysis of 11 isolates of Salmonella enterica serovar Agona from a 2008 outbreak in the United States associated with a commercial cereal product. Identical PFGE patterns with XbaI were resolved by digestion with BlnI. (PulseNet, unpublished.)
Restriction enzymes yielding optimum number and size range of chromosomal fragments for analysis by PFGE a
Representative intenelationships between PFGE switching intervals and DNA size a