LABORATORY PROTOCOL

PCR for plasmid-mediated colistin resistance genes,

mcr-1 and mcr-2 (multiplex)
(protocol optimized at National Food Institute, Denmark)

October 2016
Version 2

Lina Cavaco, Hanne Mordhorst, Rene Hendriksen

HISTORY OF CHANGES
Version Sections Description of change Date Approval
changed
1 New document - December Authors
2015
2 Throughout Addition of mcr-2 and October 2016 Authors
document adjustments to use Dream
Taq Green Mastermix in the
reaction
 PROTOCOL
PCR protocol for the mcr-1and mcr-2 genes
(multiplex)

Contents Page

MATERIALS ..................................................................................................................................................................... 3

PCR DETECTION OF MCR-1 AND MCR-2 (MULTIPLEX) ...................................................................................... 4

REFERENCES ................................................................................................................................................................. 5

DETAILED PROCEDURE .............................................................................................................................................. 6

COMPOSITION AND PREPARATION OF MEDIA AND REAGENTS ................................................................... 9

APPENDIX 1 (LABORATORY SAFETY) .................................................................................................................. 10

APPENDIX 2 (EXAMPLE OF PCR SET UP) ............................................................................................................ 15

2
Materials
Equipment
• PCR thermocycler
• Pipettes for 1 μL to 1000 μL
• Electrophoresis unit
• Microwave or autoclave
• Eppendorf centrifuge
• Photo camera
• UV-transilluminator
• Water bath 50oC

Materials
• Molecular marker (Ladder 100bp)
• Ethidium bromide solution (1%)
• Staining bath
• Electrophoresis buffer TAE or TBE (see composition of media and reagents)
• Eppendorf tubes
• Tips (filter) for pipettes 1 μL to 1000 μL
• Agarose
• Primers
• Dream Taq Green PCR Master Mix (2X) (Thermo Fisher)
• DNA template (boiling lysates)
• TE buffer (Tris:EDTA 10:1)
• TrisHCl buffer
• Crushed ice
• Mineral oil (if necessary)

Safety
Carry out all procedures in accordance with the local codes of safe practice.
For staining the DNA, ethidium bromide is used. This dye is carcinogenic. Therefore
gloves and proper clothes should be worn (appendix 1: laboratory safety).
Visualisation of the stained DNA is done by use of UV transilluminator. UV light is harmful
for skin and eyes. Therefore proper protection (facemask, glasses) should be worn
(Appendix 1: laboratory safety).

3
PCR detection of mcr-1 and mcr-2 (multiplex)
The mcr-1 gene was described by Liu et al. who published their findings in a Lancet article in the
end of 2015. This gene encodes phosphoethanolamine transferase enzyme family, with
expression in E. coli resulting in the addition of phosphoethanolamine to lipid A and in this way
confers resistance to colistin. A similar gene, mcr-2 was described in Belgian isolates by Xavier et
al, 2016 and published in Eurosurveillance.

This protocol has been modified by adding the mcr-2 reaction to the previous protocol optimized at
Statens Serum Institute (SSI, 2015) and by using a different Taq polymerase containing
mastermix.

Control strains

E. coli carrying mcr-1 (ID: 2012-60-1176-27)

E. coli containing mcr-2 (ID: KP37) obtained from Xavier et al.

DNA extraction
The template DNA used consisted of boiling lysates prepared from the strains. A brief description:
a loopful of culture was suspended in 100 µl of sterile TE buffer, boiled 10 min at 100ºC,
centrifuged 5 min at 6000 G. For use as template in the PCR, the DNA supernatant was further
diluted at 1:10 in TrisHCl buffer.

Primers

Target gene Primer sequences Reference

CLR F 5'-CGGTCAGTCCGTTTGTTC-3'
mcr-1 (35-343) Liu et al., 2015
CLR R 5'-CTTGGTCGGTCTGTAGGG-3'
MCR2 IF 5'- TGTTGCTTGTGCCGATTGGA-3'
mcr-2 (494-1060) Xavier et al., 2016
MCR2 IR 5'-AGATGGTATTGTTGGTTGCTG-3'

Reaction mix
Prepare the following mix in a microcentrifuge tube (for a 25µL reaction; see the example of PCR
set up). Additionally, prepare a blank reaction without template DNA as negative control. This
version of the PCR protocol is optimized for the Dream Taq Green PCR Master Mix (2X)
(https://tools.thermofisher.com/content/sfs/manuals/MAN0012704_DreamTaq_Green_PCR_Maste
rMix_K1081_UG.pdf) which can be replaced by another polymerase, although the protocol might
need some optimization to adjust for the particular conditions at your laboratory in which we can
give you some assistance.

4
Dream Taq PCR Master Mix* 12.5 µL
Primer Mix** 2 µL
DNA template 2 µL
Water up to 25 µL

*This PCR at the EURL-AR laboratory is optimized for the Dream Taq Green PCR Master Mix
(Thermo Fisher) which already contains MgCl2.
**The primer mix contains 2µM of each primer in water or TE buffer

Conditions for the PCR

94ºC 15 min + 25X (94ºC 30 sec + 58ºC 90 sec + 72ºC 60 sec) + 72ºC 10min.

References
Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, Doi Y, Tian G, Dong B, Huang X, Yu LF,
Gu D, Ren H, Chen X, Lv L, He D, Zhou H, Liang Z, Liu JH, Shen J. Emergence of plasmid-
mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a
microbiological and molecular biological study.
Lancet Infect Dis. 2015 Nov 18. pii: S1473-3099(15)00424-7. doi: 10.1016/S1473-3099(15)00424-
7. [Statens Serum Institute, working protocol (Personal communication Frank Hansen and Henrik
Hasman)]

Xavier BB, Lammens C, Ruhal R, Kumar-Singh S, Butaye P, Goossens H, Malhotra-Kumar S.

Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli,
Belgium, June 2016. Euro Surveill. 2016;21(27):pii=30280. DOI: http://dx.doi.org/10.2807/1560-
7917.ES.2016.21.27.30280

5
Detailed Procedure Theory / comments

Preparation of the samples

1. Transfer 100 µL of TE buffer to a Make a homogeneous cell suspension in an

1.5 mL Eppendorf tube. Using a Eppendorf tube, using a loop or cotton swab.
disposable inoculation loop (white; Shake or vortex suspension just before use.
1µL), pick a loop full of bacteria Only very little cell mass is needed. If too
from a plate and transfer to the many bacteria are used, it might cause
Eppendorf tube. inhibition of the PCR.

2. Boil the suspension (or heat at TE is used because it contains EDTA that
95°C) for 5-10 minutes. binds divalent ions needed by enzymes that
would be able to degrade DNA. Since EDTA
3. Centrifuge at 6000 G for 5 min. can inhibit the PCR reaction, dilute in TrisHCl

4. Dilute the supernatant lysed DNA

10-fold in TrisHCl Boiling breaks down the bacterial cell wall and
allows release of DNA.
Make a ventilation hole in the lid of the
Eppendorf tube using a needle, alternatively
the eppendorf tubes can be capped with a
lidlock.

Preparation of the mix

1. Check the number of samples and Always prepare mix for at least 1-2 additional
calculate the amount of PCR samples (n mix = n samples + 1 or 2).
master mix needed. Use crushed ice if the PCR master mix is
prepared at temperatures above 25°C as this
2. Prepare the PCR master mix (you may affect the result.
may do it in a tray of crushed ice as
mentioned in appendix 2). If the PCR machine has no heat in the lid
use oil as a lid to avoid the mixture to vaporise
3. Aliquot the PCR master mix to the and condensate in the lid of the tubes
required number of PCR tubes (23
μL per tube).

4. Depending on the PCR machine

that is used, one drop of mineral oil
could be added.

6
Running the PCR Theory / comments

1. Add 2 μL of sample to the sample Always end the set up with the positive control
tube and close the lid. DNA to avoid contaminations of the test tubes,
causing false positive results.
2. Add 2 μL of water to the negative
control tube and close the lid For PCR a positive control and a negative
control (sterile water) should be taken along.
3. Finish the procedure by adding the
positive control DNA and close the If oil is used, make sure DNA is dispensed
lid. below the oil phase.

4. Place the tubes into the PCR

thermocycler.

5. Program the PCR thermocycler (or

select the requested program) as
mentioned in appendix 2 (Example
of PCR set-up).

6. Run the program.

Preparation of the agarose gel

1. Assemble the gel tray and make a

proper set-up.

2. Prepare a 1.5% agarose solution in Depending of electrophoresis system use TBE

TBE buffer 1X by boiling the or TAE buffer
solution a few minutes until
completely dissolved by the use of
a water bath or microwave oven.

3. Cool the agarose to 40-50oC in a

water bath and pour the agarose
into the gel tray.

4. Let the gel solidify for 15-30

minutes.

5. Prepare a staining bath containing Wear suitable protection for working with
a final concentration of 5 µg/mL ethidium bromide (gloves, clothes). Appendix
ethidium bromide. 1: laboratory safety.

7
Assembling the results Theory / comments
1. Put the gel into the electrophoresis TBE contains boric acid. See Appendix 1 for
unit and if necessary refill with safety sheet.
buffer.
The Dream Taq Green Master Mix contains
2. Load 8 µL of each PCR sample into two tracking dyes and a density reagent that
the wells of the gel. Finish off by allows the direct loading of PCR product
loading at least one molecular
marker. It is important to use a proper marker in order
to notice whether the PCR product has the
3. Replace the lid of the unit and run right size.
the gel by starting the
electrophoresis process. Electrophoresis can be done at different
voltages/amperages. Normally, 70mA seems
4. After a complete run of 30-45 to be fine. Running time depends on several
minutes, remove the lid of the unit parameters like buffer composition, resistance,
and place the gel in a staining-bath current.
for about 30 minutes. Rinse shortly
in water before visualising the gel / Be extremely careful and wear the correct
bands. protective gloves when dealing with the
ethidium bromide staining bath, please read
5. Place the gel / tray on top of the the safety precautions in Appendix 1)
UV-transilluminator.
UV light is harmful for skin and eyes. Wear
6. Visualise the results by switching proper protection (facemask) see Appendix 1,
on the UV-lamp. laboratory safety

7. Look for the presence of specific The whole PCR process is very sensitive
bands (Appendix 2: Example of towards contaminations that can affect the
PCR set up). result as false positive results. It is therefore
recommended to perform the different steps, if
possible, in different rooms e.g.:
Room 1: Preparing the PCR master mix into
tubes.
Room 2: Adding the samples to the tubes and
running the samples in the PCR thermocycler.
Room 3: Running the electrophoresis and
visualising the DNA.

8
Composition and preparation of media and reagents
Reagents can be made as described below and/or are commercially available from
companies like Invitrogen Life Technologies and Roche Applied Science.

TAE (Tris-Acetate EDTA) buffer

Working solution
• 0.04 M Tris Acetate
• 0.001 M EDTA

Concentrated stock solution (50X)

Per liter
• Tris base 242 g
• Glacial acetic acid 57.1 mL
• 0.5 M EDTA (pH 8.0) 100 mL

TBE (Tris-Borate EDTA) buffer

Working solution
• 0.089 M Tris borate
• 0.089 M boric-acid
• 0.002 M EDTA

Concentrated stock solution (5X)

Per liter
• Tris base 54 g
• Boric acid 27.5 g
• 0.5 M EDTA (pH 8.0) 20 mL

Tris-EDTA buffer (TE 10:1) 1 L (pH 8)

• 1 M Tris-HCl (pH 8) 10 mL
• 0.5 M EDTA (pH 8.0) 2 mL
• Water 988 mL

Tris-HCl buffer 1 L (pH 8)

1 M Tris-HCl (pH 8) 10 ml
Water 990 ml

Ethidium bromide (10mg/mL)

• Add 1 g of ethidium bromide to 100 mL of H2O. Stir on a magnetic stirrer for several
hours to ensure that the dye has dissolved. Wrap the container in aluminum foil or
transfer to a dark bottle and store at 4oC.
• Ethiduim bromide can be bought ready-made as a 10 mg/ml solution
 APPENDIX 1 (laboratory safety)
Safe Work Procedure ETHIDIUM BROMIDE

Use
Ethidium bromide is added to electrophoresis gels for visualisation of nucleic acids
Hazards
Class 6 – Toxic. Potent mutagen
Risk control measures
Only use ethidium bromide (EtBr) after receiving safety training (laboratory induction /
authorisation). Wear safety glasses when using ethidium bromide. Avoid skin contact;
ethidium bromide may be absorbed through the skin. Wear latex gloves, laboratory coat.
Always dispose of gloves after use. Do not touch equipment, door handles, phone,
keyboard, etc.
Weighing solid - Powder may cause irritation when inhaled - wear dust mask and use in
ventilated area. Use designated micropipette, only, when dispensing the liquid.
Engineering / Ventilation controls
Ensure access to a safety shower and eye wash in areas where ethidium bromide is used.
Preferably weigh the solid in a fume hood.
Storage requirements
Store in a cool, dry place away from strong oxidizing agents. Keep containers tightly
closed. Use with adequate ventilation.
First aid / Spill control procedures
Wash off immediately with copious amounts of cold water (at least 10 minutes). Ethidium
bromide is absorbed through the skin so follow the cold water washing with a thorough
washing with warm water and soap. Contaminated clothing should be removed as soon as
possible and thoroughly washed.
In case of contact with eyes, immediately flush eyes with copious amounts of water for at
least 15 minutes (eye wash).
Seek medical attention.
If the spill is on equipment, use ultraviolet light (wear appropriate eye protection) to locate
spill, then use the decontamination procedure outlined below.
Wear protective clothing.
Small spill: If in solution, absorb freestanding liquid using vermiculite or Polyzorb from Spill
Kit. Use ultraviolet light to locate spill. Follow instructions on Spill Kit.
Large Spill: Notify others in the area of spill. Evacuate area. Barricade area with tape (in
Spill Kits) to prevent entry until arrival of response personnel. Provide assistance and
information to spill clean up crew.

10
Waste
Store waste; liquid: In proper waste container
Store waste; Solid: In proper waste container

Ethidium bromide liquid disposal

1. Add 10g activated charcoal per 2.5L waste
2. Leave for 1 hour, with occasional shaking
3. Filter contents through Whatman Number 1 filter paper.
4. Filtrate may be disposed of down the sink.
5. Charcoal & paper is treated as solid hazardous waste and disposed in the EtBr Solid
Waste Bucket.
If Using ‘Green Bag’ (Bio-101 Cat. No. 2350-200):
1 For 10mg Ethidium Bromide (max) add 1 ‘Green Bag’ to the waste bottle with a magnetic
flea.
2. Place waste bottle onto a magnetic stirrer and mix the solution for 24hours.
3. Dispose of the ‘Green Bag’ in the dry Ethidium Bromide waste. The remaining solution
may be disposed of in the sink.

Staining gels
During electrophoresis, add EtBr after boiling up the agarose - let it cool down before
adding EtBr
Afterwards, soak gel in a well-marked plastic container - put name and date on container
as it is possible to re-use the staining solution.

11
 Safe Work Procedure ULTRA VIOLET SOURCE

Ultraviolet light
Ultraviolet radiation is the portion of the electromagnetic spectrum that falls in the region of
100 to 400nm. This spectrum has been divided into three regions:

A: 400nm to 315nm known as Near-UV or UV-A

B: 315nm to 280nm known as Mid-UV or UV-B
C: 280nm to 100nm known as Far-UV or UV-C

Hazards
Two categories of hazard are involved in the use of high intensity UV lamps: those
inherent in the radiation itself and those associated with operation of the lamps. All
radiation of wavelength shorter than 250 nm should be considered dangerous.
• Damage to eyes and skin caused by exposure to UV radiation. Repeated
overexposure of skin to UV has been linked with premature aging, wrinkles and
most seriously, skin cancer. Eye damage can result in corneal scarring and cataract
formation.
• Burns caused by contact with a hot UV lamp.
• Fire ignited by hot UV lamp.
• Interaction of other nearby chemicals with UV radiation.
Damage caused to apparatus placed close to UV lamp

Risks
Damage to vision is likely following exposure to high intensity UV radiation.
Who is likely to be injured?
The user or anyone exposed to the UV light as a result of faulty procedure. Injuries may be
slight to severe.

Control measures operating precautions

Lab-coats, gloves and safety glasses or other appropriate eye/skin protection such as UV
protective glasses or a UV protective face shield must be worn.

Reactions using UV lamps: external irradiation sources

• These operations must never be attempted by an untrained person.
• These operations must never be attempted by a single person.
• These operations must never be attempted out of normal working hours.
• Use of UV lamps must be carried out in a fume hood with boarded up windows.
• As far as possible, the UV source should be contained in a closed radiation box.
• The fume hood sash must remain closed while the UV lamp is switched on.

12
 • The fume hood may contain only the UV lamp and associated apparatus and
chemicals. No other chemicals are to be stored in the fume hood and no other
reactions are to be performed in the fume hood.
• Reaction vessels containing flammable solvents must be at least 20 cm away from
the lamp to avoid excessive heating.
• Flammable equipment (e.g. rubber/plastic tubing) must be positioned at least 10 cm
away from the lamp.
• After the UV lamp is switched off, unless the reaction mixture requires immediate
attention, the fume hood sash should remain closed for 30 minutes to allow the UV
lamp to cool.

Reactions using UV lamps: low/medium pressure Hg lamps in an immersion well

• These operations must never be attempted by an untrained person.
• These operations must never be attempted by a single person.
• Low/ Medium pressure lamps are to be used ONLY in approved, water-cooled
immersion well apparatus.
• The UV lamp power supplies must incorporate an electrical cutout that activates in
the event of disruption to cooling water.
• The UV lamp must not be switched on until:

• The glassware is shrouded in Al foil

• The immersion well set-up is shielded by the appropriate metal case

• The boarded up fume-hood doors are closed

• The UV lamp must NEVER be switched on/connected outside of the shrouded
immersion well apparatus.
Training
For the use of high intensity UV sources, new users must be trained by another member of
the laboratory who, in the opinion of the member of staff in charge of the laboratory, is
sufficiently competent to give instruction on the correct procedure. Newly trained users
should be overseen for some time by a competent person.
Emergency Procedures
UV exposure: Act according to local procedures and provide first aid to the injured. If
necessary prepare a report for working accidents.
Burns: Act according to local procedures and provide first aid to the injured. If necessary
prepare a report for working accidents.

13
 Hazard sheet BORIC ACID
Synonyms: Boric Acid, MB Grade (1.12015); Boron; Boricacidhighpurity; Boricacidwhitextl

Molecular Formula: H3BO3

Formula Weight: 61.83
Registry number: 10043-35-3

Registry number: 10043-35-3

Density: 1.43
Melting point: 169 °C

Hazard Symbol

Toxic

Risk Description
R60 May impair fertility.

Safety Description
S45 In case of accident of if you feel unwell, seek medical advice immediately (show the
label where possible).
S53 Avoid exposure - obtain special instructions before use.

IR
Analysis Result

Miscellaneous 545 (29.51); 641 (30.64); 813 (26.79); 1193 (21.05); 1456 (17.55);
3215 (15.75)

14
 APPENDIX 2 (Example of PCR set up)
PCR Date: Initials:
Primers Forward: CLR F 5'-CGGTCAGTCCGTTTGTTC-'3
MCR2-IF 5'-TGTTGCTTGTGCCGATTGGA-'3
Primers Reverse: CLR R 5'-CTTGGTCGGTCTGTAGGG-'3
MCR2-IR 5'-AGATGGTATTGTTGGTTGCTG-'3
DNA polymerase: DreamTaq™ Green PCR Master Mix
PCR product: mcr-1: 309 bp
mcr-2: 567 bp
Remarks: Plasmid-meditated colistin resistance
Use 2µl boiling lysate as template

Positive controls: mcr-1: E. coli 2012-60-1176-27

mcr-2: E. coli KP37

1. 15 min at 94 °C
Number of samples 1 8
PCR H2O 8,5 68 2. 25 cycles
2xGreen PCR Master Mix 12,5 100 30 sec at 94 °C
90 sec at 58 °C
60 sec at 72 °C
Primers F - 0,5 µl of each 1 8
3. 10 min at 72 °C
Primers R - 0,5 µl of each 1 8
4. hold at 4 °C
Total volume 23 184

M: 100 bp Ladder plus

1 Sample 1
2 Sample 2
3 mcr-1 positive control
4 mcr-2 positive control
5 Sample 3
6 mcr-1 and 2 positive control mix
7 mastermix
8
9
10
11
12

15