pulse field gel electrophoresis (pfge) explanation bob wickert, ms, mt(ascp) molecular...
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Pulse Field Gel Electrophoresis (PFGE) Explanation
Bob Wickert, MS, MT(ASCP)
Molecular Microbiologist
Nebraska Public Health Laboratory
E.coli O157:H7
Targetregionfor PFGE
Principle: Strain diversity
Foreign DNA
Host DNA
New Host DNA
Insertion event
Principle: Strain diversity
Host DNA
New Host DNA
Deletion event
Excised fragment
Principle: Strain diversity
Host DNA
New Host DNA
Rearrangement event
Principle: PFGE SubtypingPrinciple: 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).
Practice
24 hour culture
Cell suspension
abs ~1.8
Make agarose plugs
Lyse cells and wash plugs
Practice
Restrict DNA in plugsSlice 2mm piece of plug
Load slices onto comb
Pour gel and remove comb
Electric current 18-20 hours
buffer 14 Celectrodes
The End Result
The Analysis Process: Normalization
The Analysis Process: Making Comparisons
Key
IsolatDate
Serotype
PFGE-XbaI-pattern
REF 1784
2000-07-12
E. Coli O157:H7
x.0055
REF 1823
2000-07-07
E. Coli O157:H7
x.0055
544.38
445.20
348.09
302.10
272.09257.50242.26219.29
174.57155.51
122.80
99.16
66.4060.8052.5045.0637.5132.87
546.15
444.60
349.53
303.89
272.09257.50243.18221.80
179.37159.57
123.53
101.29
68.3062.2552.8246.0439.7035.70
Similarity: 100.00%
Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-98.3%]
Unmatched bands: 0
-Gray boxes indicate unique PFGE patterns-Colored boxes indicate PFGE patterns seenmore than once. Numbers in box indicate PFGE pattern as noted in state O157 PFGEepidemiology report
1413121110 9 8 7 6 5 4 3 2 1
Jan Feb Apr Jun Jul Aug Sep Oct Nov
2
55
55
2
48
48
48
E.coli O157:H7Surveillance
Nebraska, 2003
E.coli O157:H7Surveillance
Nebraska, 2003
# Cases
2
2
153
55
48
55
2
2
48
2
48 153
5555
2
2
82 82
75
75
Nebraska Nebraska E. coliE. coli Database 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
• 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
• 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
