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Pulse Field Gel Electrophoresis (PFGE) Explanation

Pulse Field Gel Electrophoresis (PFGE) Explanation. Bob Wickert, MS, MT(ASCP) Molecular Microbiologist Nebraska Public Health Laboratory. Target region for PFGE. E.coli O157:H7. Principle: Strain diversity. Insertion event. Foreign DNA. Host DNA. New Host DNA.

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Pulse Field Gel Electrophoresis (PFGE) Explanation

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  1. Pulse Field Gel Electrophoresis (PFGE) Explanation Bob Wickert, MS, MT(ASCP) Molecular Microbiologist Nebraska Public Health Laboratory

  2. Target region for PFGE E.coli O157:H7

  3. Principle: Strain diversity Insertion event Foreign DNA Host DNA New Host DNA

  4. Principle: Strain diversity Deletion event Host DNA Excised fragment New Host DNA

  5. Principle: Strain diversity Rearrangement event Host DNA New Host DNA

  6. Principle: PFGE Subtyping • Strain diversity (insertions, deletions & rearrangements) give rise to changes in DNA fragments. • Subsequent generations of bacteria retain the parental DNA pattern until another diversity event (clonality).

  7. Cell suspension abs ~1.8 24 hour culture Lyse cells and wash plugs Make agarose plugs Practice

  8. Slice 2mm piece of plug Restrict DNA in plugs Load slices onto comb Pour gel and remove comb Practice

  9. Electric current 18-20 hours buffer 14 C electrodes

  10. The End Result

  11. The Analysis Process: Normalization

  12. The Analysis Process: Making Comparisons

  13. 14 13 12 11 10 9 8 7 6 5 4 3 2 1 # Cases E.coli O157:H7SurveillanceNebraska, 2003 2 82 82 2 48 55 -Gray boxes indicate unique PFGE patterns -Colored boxes indicate PFGE patterns seen more than once. Numbers in box indicate PFGE pattern as noted in state O157 PFGE epidemiology report 48 55 55 48 55 55 2 2 2 2 2 2 55 2 75 48 153 75 48 153 48 Jan Feb Apr Jun Jul Aug Sep Oct Nov

  14. Nebraska E. coli Database • 308 isolates typed since 1999 • 170 pfge patterns • 138 (44%) patterns observed only once • Most common pattern (x.0002) seen 32 times = 10% database

  15. A match does NOT mean the cases are DEFINATELY related A non-match does not mean that the cases are definitely NOT related. Microbial Subtyping: What it doesn’t mean

  16. A match means the cases are MORE LIKELY to have a common source than if they didn’t match A non-match means the cases are LESS LIKELY to have a common source than if they did match Microbial Subtyping: What it does mean

  17. Any questions??Contact informationrwickert@unmc.edu559-2123

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