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Pulse Field Gel Electrophoresis (PFGE) Explanation Bob Wickert, MS, MT(ASCP) Molecular...
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Transcript of Pulse Field Gel Electrophoresis (PFGE) Explanation Bob Wickert, MS, MT(ASCP) Molecular...
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