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Detailed laboratory protocol for characterisation of B. Pertussis using Pulsed field gel electrophoresis (PFGE).
This PFGE method has been developed by our French colleagues at the Institut Pasteur and has been first published in “Mooi, F. R., H. Hallander, C. H. Wirsing von König, B. Hoet, and N. Guiso. 2000. Epidemiological typing of Bordetella pertussis isolates: recommendations for a standard methodology. Eur J Clin Microbiol Infect Dis 19:174-181”. This method has been performed at The Swedish Institute for Infectious Disease Control and was the basis for the reference system published in 2004, “Advani, A., D. Donnelly, and H. Hallander. 2004. Reference System for Characterization of Bordetella pertussis Pulsed-Field Gel Electrophoresis Profiles. J Clin Microbiol 42:2890-7”.
Reagents:
0.5 X TBE buffer pH 8.3
1 X TE buffer pH 7.5
0.5 mol/l EDTA pH 8.0
Lysis buffer (0.5 mol / l EDTA pH 8.0, sarkosyl 1 %)
PBS pH 7,47
Sterile water, Octaviaâ. REF 88 33 14
EtBr (10mg/ml)
Materials:
55 oC incubator
36 oC and 37 oC incubator
Petri dish.
2 scalpels
low melting agarose (SeaPlaqueâagarose, FMC BioProducts, Cat. No. 50100. 125 g)
SeaKemâGTGâAgarose, Cambrex. Cat. No. 50070. 125 g.
Proteinase K (fungal) 20 mg / ml, (Cat. No.25530-031, 1 g(>20units / mg) Invitrogen, Germany)
XbaI, Pharmacia Biotech, 15000 U, Cat. No. E1093ZH (0.1 % BSA & 10 X M buffer are supplied with enzyme)
Low Range PFG Marker (Cat. No.N0350S, New England Biolabs).
T41 calibrator
Transpipette 1 ml Sarstedt No./ REF 86.1180
Transpipette 3.5 ml Sarstedt No./ REF 86.1172
PP-Test tubes, 50 ml sterile. Cat. No. 227261. CELLSTARâ, Greiner bio-one
10 ml centrifuge tube with yellow screw cap (Sarstedt Cat. No. 62.9924.284)
1.5 ml micro tubes, Sarstedt, No./ REF 72.690 white)
1.5 ml Cuvettes (Plastibrand, GMBH, Germany Cat. No. 759015)
white loop (Servant, Cat. No. 0016, 1 µl loop)
Safe-Lock Tubes 2.0 ml, Eppendorf, Cat. No. 0030 120.094
Blue loops (Servant, Cat. No. 0018, 10 µl loop
2.0 ml Sarstedt tube. Cat. No. 72.694.006
Spectrophotometer
Fridge
Freezer
A DR III contour-clamped homogeneous electric field apparatus with a cooling module and variable speed pump (Bio-Rad Laboratories CA USA)
Gel Doc 2000 (Bio-Rad Laboratories CA USA)
BioNumerics v3 software (Applaied Maths Sint-Martens-Latem, Belgium)
DAY 1
E.g. starting on a Monday, based on 20-tooth comb.
Turn on 55 oC incubator.
PFGE
-Inoculate 1ml 1 X PBS buffer in 10ml centrifuge tube with yellow screw cap with a white loop full of bacteria.
-Vortex.
Measure optical density (OD)
-Measure optical density at 650 nm.
-Add 100 µl bacterial suspension to labelled cuvettes containing 900µl 1 X PBS buffer.
-Use 1 ml 1 X PBS as blank.
-OD should be adjusted to 0.8.
-Add required amount 1 X PBS to each tube to adjust the OD.
Preparing agarose plugs
-Prepare 10mls of 1 % low melting agarose in 0.5 X TBE buffer.
-Boil agarose in 100 ml conical flask and then transfer agarose to 10 ml yellow top tube.
-Add washings from conical flask, around 1 ml warmed deionised water, to bring volume up to 10 ml.
-Vortex tube.
-Place in 55 oC incubator.
-Equilibrate agarose gel to 55 oC for at least one hour.
-Tape bottom of plug moulds and number accordingly.
-Prepare plug moulds on ice.
-Prepare a mix of 150 µl bacterial Suspension and 150 µl low melting agarose in 1.5ml Eppendorf micro tubes.
-Fill desired amount of plug moulds for each sample.
-Leave to set on ice for at least 20 minutes.
-Transfer plugs to 2.0 ml tubes containing lysis buffer (lysis buffer and proteinase K) and incubate overnight in a shaking incubator at 55 oC.
Preparing Proteinase K
Use 1 mg / ml proteinase K per plug.
E.g. To treat 1 plug with Proteinase K: Incubate in 15 µl Proteinase K (20 mg / ml) + 285 µl lysis buffer.
Incubate 300 µl of this solution per plug per tube.
Or- for 10 plugs: - Mix 150 µl PK and 2.85 ml lysis solution in a 50 ml Falcon tube.
Dispense 300 µl of solution to each of 10, 2 ml round bottom tubes.
Push plugs into lysis solution in labelled tubes.
1 plug: 15 µl PK + 285 µl lysis buffer
10 plugs: 150 µl PK + 2.85 ml lysis buffer
20 samples: 300 µl PK + 5.7 ml lysis.
40 samples: 600 µl PK + 11.4 ml lysis solution.
Incubate plugs overnight at 55 oC in shaking incubator.
DAY 2
Turn on 55 oC incubator.
Washing plugs
-Remove Proteinase K treated plugs from 55 oC incubator and cool plugs on ice water for at least 10 minutes.
-Prepare a set of Falcon tubes for washing of plugs. Label tubes 1-20 etc.
-Transfer plugs to labelled Falcon tubes.
-Wash plugs with pre-warmed 1 X TE buffer at 55 oC in a shaking incubator.
-Buffer can be pre-warmed Tuesday morning in microwave or left in 55 oC incubator Monday evening.
-Add 7.5ml of 1 X TE buffer pre-warmed to 55 oC to each tube. (55 oC to inactivate PK).
-7.5 ml 1 X TE buffer per plug
-Place tubes on shaker at 55 oC for at least one hour.
-Important to wash off and remove all buffer in between each washing step.
-Make sure plug is at bottom of tube after buffer has been decanted between each washing step (don’t want to carry over any residual PK)
-Between each washing step cool plugs on ice water for 10 minutes.
-Decant buffer.
-Repeat wash with fresh buffer at 55 oC.
-Perform a 3rd and final wash at room temperature.
XbaI treatment
Digest one plug slice with 50U XbaI per 150µl volume.
E.g. for digestion of 10 plugs prepare enzyme mix as follows:
|
N |
µl H20 |
µl 100x0.1% BSA |
µl 10xM Buffer |
µl 50U/µL Enzyme |
|
10 |
1325 |
15 |
150 |
10 |
-Label appropriate number of 1.5 ml micro tubes.
-Add 15 0µl enzyme mix to each tube.
-Digest T41 along with samples to be set on a 20-tooth comb.
-Take one plug from falcon tube and place in a petri dish.
-Using 1st scalpel slice off one small rectangular plug and place under surface of enzyme mix -in correctly labelled tube.
-Using a 2nd separate scalpel, place remaining piece of PK digested plug into EDTA storage tube.
-Label appropriate number of 2.0ml screw top sarstedt tubes and add 750µl 0.5 mol/l EDTA.
-Proteinase K treated plugs are stored in EDTA at 4 oC)
-Incubate plug slices at 37 oC overnight. (Incubation time is around 16 hours)
DAY 3
Running the gel.
Turn on 55 oC incubator.
-Remove enzyme treated plug slices from 37 oC incubator.
-Remove enzyme reaction mix from tubes with 1ml Transpipette.
-Add 250 µl 0.5 mol/l EDTA to each tube to stop reaction.
-Tap tube slightly to mix.
-Leave at room temperature (can leave on shaker) for 15 minutes then store at 4oC (for 2-3 weeks).
-Remove EDTA from tubes.
-Add 1.3 ml 0.5 X TBE.
-Tube no. 11 always contains T41 calibrator.
-Tubes no. 2, 10 & 19 always contain the low range PFG marker.
-The low range marker is buffered on the day the gel is to be run.
-Prepare low range marker in 0.5 X TBE.
-Cut 3 slices of the Low Range Marker approximately 1 mm in thickness.
-Low range marker can be pre-cut and left in 0.5 X TBE buffer in fridge in 2.0 ml tube for a week in advance.
Incubate plugs at 4 oC for one hour.
Mix by inverting several times every 15 minutes.
PREPARING THE GEL
-Prepare 120 ml 1 % agarose in 0.5 X TBE (dissolve 1.2 g SeaKem Agarose in 120 ml 0.5 X TBE).
-Boil with stirring for one minute.
-Calibrate gel at 55 oC for at least one hour.
PREPARE PULSED FIELD APPARATUS
-Prepare pulse field apparatus, balance base, add gel frame.
Add 2.550 ml of 0.5 X TBE to chamber.
Start pump with speed set to 70
Start cooler.
Running temperature should be 14 oC.
Prepare frame to set gel.
Screw frame together including metal base plate.
When balanced cover frame to keep off dust.
PLACING PLUG SLICES ON COMB
-Place 20 toothcomb on bench.
-Place each plug at end of each appropriate tooth on comb.
-Remove excess buffer.
-Add one drop of agarose to each tooth to stick plug to tooth.
-Leave for 4 minutes and then place comb in gel frame.
-Slowly pour the 120 ml gel into frame and set gel for 60 minutes.
-Unscrew from frame and remove excess pieces of agarose from the edges and set gel between frame in PF buffer chamber.
Programing PF apparatus: Xba1, -
Sec. V/m hours
Block 1: 5-6 5.5 16
Block 2: 8-35 5.5 24.
DAY 5
Staining and recording of PFGE image
-50 µl EtBr (10mg/ml) is added to 500 ml deionised water in a staining tray.
-Stain gel for 30 minutes.
-Rinse gel in H2O prior to destaining.
-Destain gel in at least 2 L of deionised water for 1 hour with gentle shaking.
-Record image on for example, Gel Doc 2000 apparatus, (F6, 7 seconds).
-Save as .tif file.
-Upload .tif file to BioNumerics and analyse.
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