PM 7/100(1): Rep-PCR tests for identification of bacteria

Errata

This article is corrected by:

  1. Errata: Erratum Volume 42, Issue 1, 166, Article first published online: 19 April 2012
  2. Errata: Erratum Volume 44, Issue 1, 103–104, Article first published online: 25 March 2014

Abstract

Specific scope

This standard describes how to perform rep-PCR tests for identification of bacterial isolates.

Specific approval and amendments

Approved as an EPPO Standard in 2010-09.

Introduction

Rep-PCR genomic fingerprinting makes use of DNA primers complementary to naturally occurring, highly conserved, repetitive DNA sequences, present in multiple copies in the genomes of most Gram-negative and several Gram-positive bacteria (Louws et al., 1994; Rademaker et al., 1998; Watts et al., 2001). Three families of repetitive sequences have been identified, including the 35–40 bp repetitive extragenic palindromic (REP) sequences, the 124–127 bp enterobacterial repetitive intergenic consensus (ERIC) sequence, and the 154 bp BOX element (Versalovic et al., 1994). These sequences appear to be located in distinct, intergenic positions around the genome. Primers designed from these sequences can, after PCR amplification, distinguish distinct genomic regions located between REP, ERIC or BOX elements. The amplified fragments can be resolved in a gel matrix, yielding a profile referred to as a rep-PCR genomic fingerprint. The method presented here, yielding reproducible results over the years, is based on those published by Smith et al. (2001) and Versalovic et al. (1994).

The procedure for performing the DNA isolation and the PCR is described in the Appendix.

Acknowledgements

This test description was originally drafted by Janse J (Dutch General Inspection Service, NL).

Appendix

Isolation of DNA

  • 1 Suspend 1/3 loopful of bacteria from a 48–72 h culture on nutrient agar (NA) or yeast peptone glucose agar (YPGA) in 100 μL R/DNAse free water in a 1.5 mL Eppendorf vial (approximately 109 cells mL−1). Other suitable non-selective media can be used. Once a medium and method have been selected and proven to function in the laboratory, rep-PCR should be strictly standardized following the methods selected, to obtain reproducible results and reliable libraries of rep-PCR fingerprints.
  • 2 Vortex to acquire a homogeneous suspension
  • 3 Lyse bacteria and extract DNA, preferably using a commercially available extraction kit such as the Roche High Pure PCR Template Preparation Kit or Biorad’s Chelex 100 kit. Extraction procedures for both kits are presented in this protocol. Extraction of DNA is also possible by heating the bacterial suspension for 15 min at 95°C, followed by quickly cooling on ice and pelleting down debris by centrifugation for 5 min at 7000 g and using the supernatant.
3a) Roche High Pure PCR Template Preparation Kit

Principle

  •  Bacterial cells are lysed during a short incubation with lysozyme in proteinase K and all nucleases are inactivated by guanidine-HCl.
  •  Nucleic acids bind selectively to glass fibres in the High Pure purification filter tube.
  •  Bound nucleic acids are washed with inhibitor removal buffer in order to remove PCR-inhibitory components.
  •  Bound nucleic acids are washed with wash buffer to remove salts, proteins and other cellular contaminants.
  •  Purified nucleic acids are recovered from the glass fibre using a low-salt elution buffer.
  •  Purified DNA can subsequently be used for (rep)PCR, restriction digestion or amplified fragment length polymorphism (AFLP).

Buffers, etc.

  •  Roche High Pure PCR Template Preparation Kit (Catalog no. 1 796 828)
  •  Lysozyme solution (10 mg mL−1 lysozyme in 10 mM Tris–HCl, pH 8.0)
  •  Isopropanol
  •  R/DNAse free water

Procedure

Note: Pre-warm elution buffer to 70°C

  • 1 Pipette 200 μL extract of bacterial suspension in R/DNAse free water in a 1.5 mL vial.
  • 2 Add 5 μL lysozyme solution (10 mg mL−1 lysozyme in 10 mM Tris–HCl, pH 8.0) and incubate 15 min at 37°C.
  • 3 Add 200 μL binding buffer and 40 μL proteinase K, mix immediately and incubate 10 min at 70°C.
  • 4 Add 100 μL isopropanol and mix well.
  • 5 Pipette the sample into the upper reservoir of a combined filter tube–collection tube assembly
  • 6 Centrifuge at 8000 rpm for 1 min in a microcentrifuge.
  • 7 Discard the collection tube with flowthrough. Combine the filter tube with a new collection tube and add 500 μL inhibitor removal buffer.
  • 8 Centrifuge at 8000 rpm for 1 min.
  • 9 Discard the collection tube with flowthrough. Combine the filter tube with a new collection tube and add 500 μL wash buffer.
  • 10 Centrifuge at 8000 rpm for 1 min.
  • 11 Discard the collection tube with flowthrough. Combine the filter tube with a new collection tube and add 500 μL wash buffer.
  • 12 Centrifuge at 8000 rpm for 1 min.
  • 13 Discard the collection tube with flowthrough. Combine the filter tube with a new collection tube
  • 14 Centrifuge at 14 000 rpm for 10 s to remove residual wash buffer.
  • 15 Insert the filter tube into a clean 1.5 mL reaction tube.
  • 16 Add 200 μL elution buffer that has been pre-warmed to 70°C.
  • 17 Centrifuge at 8000 rpm for 1 min.
  • 18 Discard the filter tube. The flowthrough in the reaction tube contains the DNA.
  • 19 The DNA solution can be used directly or stored in a freezer at −20º or −80°C

Remarks

  •  Vials with lysozyme solution and proteinase K should be kept on ice to avoid diminution of enzyme activity.
  •  Lysozyme solution is stored in 1-time use portions at −20°C. Non-used solutions are discarded.
3b) Chelex 100 kit

Resuspend 1.5 g Chelex 100 (BioRad 142-2832) in 25 mL R/DNAse free water (not Tris–EDTA buffer) to make a 6% solution, which can be used directly or stored after autoclaving. A 1 mL pipette is used to aliquot the Chelex, which is maintained in suspension under agitation at moderate speed.

  • 1 Prepare 1 mL cell suspensions to between OD650 0.1–0.2 and micro-centrifuge for 5 min at 10 000 g.
  • 2 Discard the supernatant carefully using a pipette.
  • 3 Resuspend the pellet in 300 μL of Chelex suspension (see above) by vortexing, and incubate in a water bath at 56°C for 20 min.
  • 4 Vortex at high speed for 10 s. Place the microfuge tube in a heated block at 100°C for 8 min. Note: Fix the caps using a weight to avoid the caps opening (explosively) during heating.
  • 5 Vortex tubes at high speed for 10 s, and immediately chill on ice.
  • 6 Centrifuge the tube for 5 min at 14000 g. Transfer 200 μL of the supernatant carefully to a new micro-centrifuge tube.
  • 7 Use 2 μL aliquot of the supernatant as template DNA.

PCR conditions

B1) PCR conditions BOX-PCR
Primers
Reference: Smith et al. (2001) and Versalovic et al. (1994)
Forward: BOXA1R 5′-CTA.CGG.CAA.GGC.GAC.GCT.GAC.G-3′
Reverse: Forward = Reverse
Product: 100–3500 bp

BOX-PCR conditions: initial denaturation at 95°C 7 min followed by 30 cycles (94°C 1 min, 53°C 1 min, 65°C 8 min) and one final step at 65°C 16 min before cooling at 4°C.

Mastermix

For reaction volume of 25 μLPer reaction (μL)Reaction (μL)Endconc.
R/DNAse-free water17.5517.55 
Reaction buffer (10×, Invitrogen)2.502.50
MgCl2 (50 mM, Invitrogen)0.750.751.5 mM
dNTP mix (10 mM each, Promega)0.500.500.2 mM
BOX AIR (20 μM)2.502.502 μM
PlatinumTaq (5 U μL−1, Invitrogen)0.200.201 U
DNA extract2.00 
Total25.0023.00 
B2) PCR conditions REP-PCR
REP-PCR
  1. I = Inosine.

Primers
Reference: Smith et al. (2001); Versalovic et al., 1994.1
Forward: REP1R-1 5′-III ICG ICG ICA TCI GGC-3′
Reverse: REP2-1 5′-ICG ITT ATC IGG CCT AC-3′

REP-PCR conditions: initial denaturation at 94°C 1 min followed by 35 cycles (95°C 7 min, 40°C 1 min, 65°C 8 min) and one final step at 65°C 16 min before cooling at 4°C.

Mastermix

For reaction volume of 25 μLPer reaction (μL)Reaction (μL)Endconc.
R/DNAse-free water15.0515.05 
Reaction buffer (10×, Invitrogen)2.502.50
MgCl2 (50 mM, Invitrogen)0.750.751.5 mM
dNTP mix (10 mM each, Promega)0.500.500.2 mM
ERIC1R2.502.502.0 μM
ERIC22.502.502.0 μM
PlatinumTaq (5 U μL−1, Invitrogen)0.200.201 U
DNA extract2.00 
Total25.0023.00 
B3) PCR conditions ERIC-PCR
Primers
Reference: Smith et al. (2001) and Versalovic et al. (1994)
Forward: ERIC1R 5′-ATG TAA GCT CCT GGG GAT TCA C-3′
Reverse: ERIC2 5′-AAG TAA GTG ACT GGG GTG AGC G-3′

ERIC-PCR conditions: initial denaturation at 95°C 7 min followed by 30 cycles (94°C 1 min, 52°C 1 min, 65°C 8 min) and one final step at 65°C 16 min before cooling at 4°C.

Mastermix

For reaction volume of 25 μLPer reaction (μL)Reaction (μL)Endconc.
R/DNAse-free water15.0515.05 
Reaction buffer (10×, Invitrogen)2.502.50
MgCl2 (50 mM, Invitrogen)0.750.751.5 mM
dNTP mix (10 mM each, Promega)0.500.500.2 mM
REP1R-1 (20 μM)2.502.502.0 μM
REP2-1 (20 μM)2.502.502.0 μM
PlatinumTaq (5 U μL−1, Invitrogen)0.200.201 U
DNA extract2.00 
Total25.0023.00 

Electrophoresis

  • 1 Prepare a 2% agarose gel of minimum 20 cm long.
  • 2 Add 6 g agarose to 300 mL 1×TBE buffer in a 0.5–1 L flask. Melt the agarose in a microwave.
  • 3 Place the gel tray in the casting system and choose appropriate combs.
  • 4 Cool dissolved agarose under running tap water to hand-warm.
  • 5 Pour agarose solution in the 15 cm gel tray, remove air bubbles with a disposable pipette tip and place the combs.
  • 6 Clean the flask/Erlenmeyer immediately with hot water to remove residual agarose.
  • 7 Leave agarose to solidify (minimum 20 min).
  • 8 Remove the combs when gel has been formed. Clean combs carefully with hot water.
  • 9 Submerge the gel in electrophoresis unit in 1×TBE buffer.
  • 10 Mix a 6–10 μL PCR sample with 1–2 μL loading buffer on a piece of Parafilm and load the gel; load the 1 kb ladder (diluted Invitrogen 2 μL ladder and 4 μL water) in a similar amount.
  • 11 Run the gel in a cold room at 90 V (for approximately 2.5 h), constant voltage. This corresponds to 6 V cm−1, measured at the distance between the electrodes.
  • 12 Stain the gel for 40 min in an ethidium bromide solution of 0.6 mg mL−1 in approximately 400 mL 0.5 × TBE, and destain for 30 min in distilled water or in 0.5 × TBE (for less background).
  • 13 Visualize and document the bands on the gel under UV light, using suitable photographic gel documentation equipment.
  • 14 Fingerprints (band patterns) can be compared by eye and checked for similarity, but patterns can also be transformed into peak patterns and compared using a computer software program such as Bionumerics (Applied Maths NV, Belgium) or a comparable program, for more sophisticated pattern analysis and comparison of strains. These programs generally also enable the creation of rep-PCR fingerprint libraries. Identification should take place on the basis of similarity to patterns of control/reference strains analysed in the same run and in other runs, and/or with library entries.
  • 15 When using simpler image-acquisition equipment (e.g. Kodak DC290), images should be saved as TIF files with a degree of resolution adequate to enable sharing the fingerprint with other laboratories holding fingerprinting libraries or platforms such as Bionumerics for databasing and analysis of these data.

Buffers

Tris-borate-EDTA (TBE) buffer according to Sambrook & Russell, 2001

5× stock solution, 1 L
Tris base54 g
Boric acid27.5 g
0.5 M EDTA (pH 8.0)20 mL
pH 8.3 

Autoclaving or filtering by 0.22 or 0.45 μm filter delays precipitation. 10×TBE is more likely to precipitate. pH of stock solution will be around pH 8.3 (do not adjust pH). If precipitate occurs, place bottle in hot water until precipitate has dissolved.

0.5×TBE solution: 45 mM Tris-borate 1 mM EDTA

Preparation of loading buffer

Bromophenol blue (10% stock solution)
Bromophenol blue5 g
Distilled water (bidest.)50 mL
Loading buffer
Glycerol (86%)3.5 mL
Bromophenol blue solution300 μL
Distilled water (bidest.)6.2 mL

(From EU Directive 98/57/EC on Ralstonia solanacearum)

or:

0.25% bromophenol blue

40% w/v sucrose in H2O

According to Sambrook & Russell, 2001

Reference

Sambrook J. and Russell D.W. (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA

Ancillary