"CAMPYNET" PROTOTYPE STANDARDISED PROTOCOL FOR

PULSE-FIELD GEL ELECTROPHORESIS-BASED DNA TYPING OF

CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI

Prepared: July 6th, 2000; Amended December 20th, 2000

The "Campynet" PFGE Subtyping Subgroup:

Stephen L. W. On, Danish Veterinary Laboratory, Denmark

Marja-Liise Hänninen, University of Helsinki, Finland

Fiona Thomson-Carter, University of Aberdeen, Scotland

Day 1
Provide bacterial growth for isolation of
1. Subculture strains onto agar media.
DNA for typing
Grow under appropriate conditions for
1-3 days.

Day 2
Prepare initial cell suspension from which
1. Harvest cells and suspend directly
DNA will be isolated. Cold buffer restricts
into 2ml of cold Pett IV buffer:
premature cell lysis and subsequent DNA
degradation.
1 M NaCl
Standard cell OD ensures that each sample
10 mM Tris pH 8
contains approximately the same amount of
DNA
10 mM EDTA

Keep samples on ice. Adjust cell

densitites to read in the range of
Macfarland 6-7.

Important: see footnote 1

2. Melt agarose (1% chromosomal Prepares agarose for making bacterial DNA
grade agarose in distilled water) and plugs.
cool to 56E C in water bath.

Agarose:

0.2g chromosomal grade agarose (e.g.

Bio-Rad, no. 162-0135)

20ml DW

3. Use a Gilson pipette to add 0.7ml Preparation of agar plugs containing whole
warm, molten chromosomal grade bacterial cells. This stage of sample
agarose suspension to 300Fl of preparation is a key step for PFGE analysis
bacterial suspension. Mix together.and of chromosomal DNA (see 8).
dispense into 5 chambers of the mould.
Set by placing in 'fridge for ca. 10 min.

Note: other methods of pouring agar

plugs, such as using plastic syringes
as moulds, can be used at the
discretion of the laboratory

4. Remove agar blocks by inserting a Lysing of bacterial cells. This leaves only
blunt-tipped glass pipette and place DNA embedded in the solid agar matrix.
each set of 5 into a 10ml plastic tube The DNA will not shear when handled
containing 3ml ESP buffer: further and remains stable for analysis by
electrophoresis.
0.5 M EDTA

1% Sarkosyl

Add 1mg/ml Proteinase K warmed at

56EC/15 min. Just before use.
Incubate overnight at 56EC.

Note 1: many workers repeat the

lysis procedure with fresh ESP after
24 hr incubation. One of us (S. On)
has found overnight incubation to be
equally effective.

Note 2: alternative lysing procedures

may be used at the discretion of the
laboratory.

DAY 3 Removes excess enzymes and chemicals

that would interfere with restriction enzyme
analysis.
1. Decant ESP buffer from samples and
wash six times, each for at least 20 min
periods, in 2 ml of 10:1 TE (10mM
Tris-1mM EDTA) buffer. Store blocks
in ca. 1.5ml fresh TE buffer in 'fridge.
The DNA sample blocks are now ready
for PFGE analysis.

DAY 3 or 4
Prepare buffer for digestion of bacterial
2. Digestion of bacterial DNA:
DNA with restriction enzymes.

Into a standard 1ml eppendorf pipette

100Fl of enzyme buffer for enzyme
SmaI. The buffer should be as
recommended by the manufacturer. For
Amersham, thsi is 80Fl Milli Q, 10Fl
Buffer 'T' [1at 0X concn.], and 10Fl
BSA)
3. Wearing rubber gloves,carefully Prepare DNA-containing agar slice for
remove an agar/DNA block using a restriction enzyme analysis. Incubation in
glass rod. Cut a sliver ca. 1mm wide buffer equilibrates sample and makes DNA
using a clean cover slip (ensure both more amenable to digestion.
edges are straight & parallel). Use the
glass rod to place sample into sample
buffer and incubate for 1hr at room
temp.

4. Remove buffer with a pipette. Add Digestion of bacterial DNA with restriction
50Fl of buffer which contains 20 units enzyme.
of enzyme/sample:

For Amersham: SmaI: 2.5Fl Sma1, 5Fl

Buffer T, 5Fl BSA, 37.5Fl Milli Q

Incubate at relevant temperature (25-

30EC for Sma1) for 5 h.

Note: DNA can alternatively be

digested with SmaI overnight, at the
discretion of the laboratory. If so,
pre-incubation with reaction buffer
is not necessary.

DAY 4 Preparation of electrolyte buffer for

electrophoresis of samples.
Electrophoresis

1. Prepare 2.5 litres of 0.5 strength

TBE buffer using 125ml 10XTBE ()
and 2,375ml Milli 'Q').

10X Tris-borate EDTA (TBE) buffer:

108g Tris base (0.9 M)

55g boric acid (0.9 M)

40 ml 0.5 M EDTA, pH 8.0

Add Milli ´Q´grade water up to 1 L.

Sterilise by autoclaving. Discard if
precipitate is observed.

Note: If stored in cold room, allow to

stand at room temperature for ca. 1
h before adding to electrophoresis
chamber. This prevents accidental
freezing of the cooling pipe.

2. Prepare 1.4% agarose gel (1.0% if 5-6. Preparation of agarose gel for
using SeaKem Gold: see Footnote 2) electrophoretic separation of bacterial DNA
for separation of DNA fragments. fragments.
Measure 5.0ml 10XTBE (Bio-Rad) and
make up to 100ml with Milli 'Q'.
Suspend 1.4g of separation grade
agarose (e.g. Bio-Rad) in this and heat
to boiling in microwave. Cool in water
bath to 50-56EC.

3. Assemble gel mould using pre-

cleaned components and pour gel into
mould. The 23 (Pharmacia equipment)-
or 30 (Bio-Rad equipment) well-
forming comb must be inserted into the
assembly before the agarose is poured
in!

4. Remove gel well-forming comb and Loading of bacterial DNA sample for
load 22 DNA slices into wells, IN THE electrophoretic separation of restriction
ARRANGEMENT DESCRIBED fragments. The arrangement of reference
BELOW. Place slice onto the side of a and molecular weight standards has been
clean plastic scalpel blade or cover slip selected to facilitate computer-assisted
and gently manipulate into well, comparative analysis.
ensuring that each slice is straight and
adheres onto the front side of the well.

Sample order for PFGE gels:

Wells 1, 6, 11, 17, 22: Normalisation

standard, a 1:1 mix of strains CNET
068 and CNET 112

Well 12: lambda ladder

Remaining wells: unknown samples

- when using the Bio-Rad 30-well

comb, the first and last 4 wells will not
be used.

5. Gently overlay wells with molten 1% Seals samples into wells so that they remain
agarose. in place during electrophoresis.

6. Add running buffer (0.5 X TBE) to Electrophoretic separation of DNA

electrophoresis chamber and run fragments.
through pump and cooling unit (12EC)
ca. 2 hour before use. Place gel (still on
perspex unit) into horizontal
electrophoresis tank such that samples
are nearest the pump inlet. Close cover
into place and set electrophoresis
conditions on power pack and
computer.

Phase 1: 0.5-40s switch time, 22.5 h.

DAY 5

1. Switch off electrophoresis unit. Visualisation of PFGE-DNA profile.

Remove gel from base and
gently place into ethidium
 bromide (3Fl/ml: i.e. 300Fl
10mg standard solution in 1L)).
Stain for 7 min.: destain in
water for 20 min. View under
UV light & photograph.

Characteristics of the captured

image

Printed hard copies / photographs of

the final gel should adhere to the
following specifications to facilitate
computerised analysis:

A. The top of the MW marker

should always be visible.
B. B. The gel image should be
taken such that the outer edges
of the first and last samples are
8.7-9.0 cm apart in the printed
image.
C. The normalisation standard
pattern (mixture of CNET
068/112) should contain 17
bands of the following
approximate sizes (kb): 650,
500, 375, 325, 275, 200, 180,
140, 130, 100, 90, 60, 45, 30,
20, 10, 5. The bands between
ca. 5-45kb may be weak but
they should be visible.
D. The 650 kb and 5 kb fragments
(i.e. the upper and lower bands)
in standard CNET 068/112, lane
11 should be 6.0 cm apart ( 5%
deviation).

Note: it may be necessary to adjust

the running voltage to obtain the
above separation levels. Most
electrophoresis units use 6V/cm as
standard. This should be increased
or decreased as required to obtain
the results given for the
normalisation standard CNET
068/112. Also see Footnote 2.

2. Pump old buffer out from Cleaning of apparatus. Clean equipment is

electrophoresis chamber. Rinse with ca. good equipment!
2.5 L Milli 'Q'. Drain, wipe with an
ethanol-soaked cloth and wipe dry.

FOOTNOTE 1: THE USE OF FORMALDEHYDE TO DEACTIVATE DNAse.

For DNAse-producing strains, the above protocol needs to be supplemented with a step
that involves the pre-treatment of bacterial cells with formaldehyde to deactivate the
Dnase. This has not been suggested as a standard method for the following reasons:

1. Formaldehyde is extremely toxic and must be carefully handled in a fume cabinet.

2. In the experience of the members of the PFGE Subgroup, relatively few strains
(<10%) require the procedure.
3. Formaldehyde treatment invariably causes increased degradation of genomic
DNA. The quality of the PFGE profile is consequently diminished.
4. Formaldehyde treatment adds time to the preparation procedure.

For strains requiring formaldehyde pretreatment to deactivate extracellular DNAse, the

following protocol for day 2 is recommended. All other steps are as stated above.

All procedures involving formaldehyde must be performed in accordance with local

safety regulations.
Day 2
Prepare initial cell suspension from
1. Harvest cells and suspend directly
which DNA will be isolated. Cold
into 2ml of cold Pett IV buffer:
buffer restricts premature cell lysis
and subsequent DNA degradation.
1 M NaCl
Standard cell OD ensures that each
10 mM Tris pH 8
sample contains approximately the
same amount of DNA
10 mM EDTA

Keep samples on ice. Adjust cell

densitites to read in the range of
Macfarland 6-7.

2. Aliquot 1ml suspension into an Formaldehyde deactivates bacterial

Eppendorf tube and add 100Fl 37% DNAse activity that may occur in
formaldehyde solution. Incubate at some strains. If left untreated sample
room temperature for 1 h. DNA degrades and cannot be
analysed.

3. Centrifuge the suspension for 10 Remove formaldehyde, which would

min. at high speed. Wash three times interfere with subsequent procedures.
in 1ml Pett IV buffer.

4. Resuspend washed cell pellet in Prepare bacterial cells for embedding

600Fl Pett IV buffer. in agarose (see 7).

5. Warm bacterial suspension to 37EC Prevents premature solidification of

by placing in water bath for a few agar plug (see 7).
minutes.
6. Seal base of agar plug mould with Prevents leaking of agar/bacterial
tape. suspension from sample mould!

7. Melt agarose (1% chromosomal Prepares agarose for making bacterial

grade agarose in distilled water) and DNA plugs.
cool to 56E C in water bath.

Agarose:

0.2g chromosomal grade agarose (e.g.

Bio-Rad, no. 162-0135)

20ml DW

8. Use a Gilson pipette to add 0.7ml Preparation of agar plugs containing

warm, molten chromosomal grade whole bacterial cells. This stage of
agarose suspension to 300Fl of sample preparation is a key step for
bacterial suspension. Mix together.and PFGE analysis of chromosomal DNA
dispense into 5 chambers of the mould. (see 8).
Set by placing in 'fridge for ca. 10 min.

Note: other methods of pouring agar

plugs, such as using plastic syringes
as moulds, can be used at the
discretion of the laboratory

9. Remove agar blocks by inserting a Lysing of bacterial cells. This leaves

blunt-tipped glass pipette and place only DNA embedded in the solid agar
each set of 5 into a 10ml plastic tube matrix. The DNA will not shear when
containing 3ml ESP buffer: handled further and remains stable for
analysis by electrophoresis.
0.5 M EDTA

1% Sarkosyl

Add 1mg/ml Proteinase K warmed at

56EC/15 min. Just before use.

Incubate overnight at 56EC.

Note 1: many workers repeat the
lysis procedure with fresh ESP after
24 hr incubation. One of us (S. On)
has found overnight incubation to be
equally effective.

Note 2: alternative lysing procedures

may be used at the discretion of the
laboratory.

FOOTNOTE 2: THE USE OF SEAKEM GOLD AGAROSE FOR

STANDARDISED PFGE-DNA TYPING OF CAMPYLOBACTER

SeaKem Gold (hereafter SKG: BioWhittaker Molecular Applications, Rockfield, USA) is

used as the standard agarose by the CDC in the "PulseNet" network for typing E. coli and
other enteric pathogens. The separation qualities of SKG are radically different from that
of most other commerical agaroses used for PFGE. However, one of us (S. On) has
determined that results with SKG can be obtained that are wholly compatible with the
Campynet standards described above that have been obtained using standard PFGE
agarose. There should in principle be no difference from comparing results obtained
between SKG and other agaroses provided one uses 1% SKG instead of 1.4% standard
agarose, and alters the running voltage accordingly.

As an example, S. On found the following running conditions yielded equivalent results.

Bio-Rad Pulsed Field Certified Agar (i.e. standard quality): 1.4% agar, 6.6V

SeaKem Gold: 1.0% agar, 5.0V.

It is recommended that you do not deviate from the stated agar concentrations when
working to Campynet standards. EITHER 1.4% standard grade, or 1.0% SKG grade,
should be used since these give equivalent results when correctly calibrated. The voltage
parameters may however differ from those stated above and it is important to attain the
characteristics of the standard (see "Day 5: characteristics of the captured image" above).
This may take some time to attain but it is achievable.