Pulse Field Gel Electrophoresis (PFGE)

Explanation

Bob Wickert, MS, MT(ASCP)

Molecular Microbiologist
Nebraska Public Health Laboratory
E.coli O157:H7

Target
region
for
PFGE
Principle: Strain diversity
Insertion event

Foreign DNA

Host DNA

New Host DNA

Principle: Strain diversity
Deletion event

Host DNA

Excised fragment

New Host DNA

Principle: Strain diversity
Rearrangement event

Host DNA

New Host DNA

 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).
 Practice

Cell suspension
abs ~1.8

24 hour culture

Lyse cells and wash

plugs
Make agarose plugs
 Practice
Slice 2mm piece of
Restrict DNA in plugs plug

Load slices onto comb

Pour gel and remove comb

 Electric current 18-20 hours
electrodes buffer 14 C
The End Result
The Analysis Process: Normalization
The Analysis Process: Making Comparisons

Key REF 1784 REF 1823

IsolatDate 2000-07-12 2000-07-07
Serotype E. Coli O157:H7 E. Coli O157:H7
PFGE-XbaI-pattern x.0055 x.0055

546.15 544.38

444.60 445.20

349.53 348.09

303.89 302.10
272.09 272.09
257.50 257.50
243.18 242.26
221.80 219.29
179.37 174.57
159.57 155.51
123.53 122.80
101.29 99.16

68.30 66.40
62.25 60.80
52.82 52.50
46.04 45.06
39.70 37.51
35.70 32.87
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
# Cases
14
13
E.coli
E.coli O157:H7
O157:H7
12 Surveillance
Surveillance
11
10
Nebraska,
Nebraska, 2003
2003 2 82 82

-Gray boxes indicate unique PFGE patterns 2 48 55

9 -Colored boxes indicate PFGE patterns seen
more than once. Numbers in box indicate 48 55 55
8 PFGE pattern as noted in state O157 PFGE
epidemiology report 48 55 55
7
6 2 2 2

5 2 2 2

4 55 2 75

3 48 153 75

2 48 153

1 48

Jan Feb Apr Jun Jul Aug Sep Oct Nov

 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
Microbial Subtyping:
What it doesnt mean

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 does mean

A match means the cases are

MORE LIKELY to have a common
source than if they didnt match
A non-match means the cases are
LESS LIKELY to have a common
source than if they did match
 Any questions??

Contact information

rwickert@unmc.edu
559-2123