SOP: Isolation and identification of priority verotoxigenic Escherichia coli (VTEC) in foods

GIFSQ — Global Institute for Food Safety & Quality

Principle

The method is applicable to the recovery and identification of priority verotoxigenic Escherichia coli (VTEC) — that is, VTEC having a high rate of association with food-borne illness. It can be used to determine compliance with Sections 4 and 7 of the Food and Drugs Act and/or other relevant federal regulations. It has been validated for use in the food category raw meats, the food type fermented (belonging to the food category of ready to eat meat and poultry), and the food items leafy greens and sprouts (belonging to the raw and raw processed food type of the fruits and vegetables food category).

Note: While this method is only approved for certain food products, as listed above, it is assumed that this method could be used with other foods. To ensure the method is fit for purpose for commodities outside the application, it is imperative that other commodities be properly validated following the criteria in the Compendium of Analytical Methods, so that the Application section can be expanded to include these new foods if the data comply with the Microbiological Methods Committee requirements.

VTEC strains possessing key virulence factors and belonging to the O serogroup family O157, O26, O103, O111 and O145 are recovered from foods by enrichment culture techniques incorporating a simple polymerase chain reaction (PCR) as an aid in the isolation of VTEC colonies, followed by characterization of presumptive isolates using a multiplex PCR and cloth-based hybridization array system (CHAS) targeting key virulence and O serogroup-specific markers.

Apparatus

Equipment

Biological safety cabinet (BSC)
Stomacher, filtered stomacher bags
Chiller block for 0.2 mL or 0.5 mL PCR tubes
Filtration apparatus (VWR Cat. no. 70690-276) with vacuum (the method includes an illustration of the filtration apparatus setup; see the note under Procedure)
Freezer capable of maintaining -20 °C
Heat block capable of accommodating 1.5 mL microcentrifuge tubes and maintaining a temperature of 100 °C (or boiling water bath)
Incubators capable of maintaining 35 °C, 42 °C, and 45 °C
Microcentrifuge, capable of spinning 1.5 mL tubes at 10,000 x g
Microcentrifuge, small (“pop centrifuge”)
Micropipettors, adjustable to cover range of volumes from 1.0 to 1000 µL
Photodocumentation system
Refrigerator capable of maintaining 4 °C
Thermal cycler equipped with a heated lid
UV cross-linker that can be adjusted to specified settings
Vortex mixer
FlashGel electrophoresis apparatus, or equivalent

Materials

Polypropylene tubes, 50 mL
Gloves, disposable
Ice and bucket
Screw-cap and regular 1.5 mL microtubes, nuclease-free, sterile
Microtube racks
PCR tubes (0.2 mL or 0.5 mL), nuclease-free, sterile
Petri dishes, sterile
Pipettes, 10 mL
Pipettor tips, filtered, nuclease-free, sterile
Pre-cut polyester cloth (DuPont Sontara 8100) strips bearing a printed sample location grid with 10 cells. These strips can be obtained from the Ottawa Laboratory (Carling) of the CFIA (Ottawa, Ontario, Canada) or purchased directly from Daves Custom Promotions Inc. (Ottawa, Ontario, Canada). No substitutions permitted.
Racks, cold, to accommodate 0.2 mL, 0.5 mL, and 1.5 mL tubes
Forceps
Wash bottles
Aluminium foil
Test tubes, sterile (10 mL capacity)

Reagents

Enrichment and strain isolation

Cefsulodin sodium salt hydrate
InstaGene Matrix (Bio-Rad, Cat. no. 732-6030)
0.1% (w/v) Peptone water
Rainbow Agar dehydrated media (Biolog, Inc., Cat. no. 80102)
Sodium chloride (NaCl), sodium phosphate dibasic (Na₂HPO₄)
Modified tryptone soya broth (mTSB, Oxoid CM0989)
Vancomycin hydrochloride
Brain Heart Infusion (BHI) agar or Nutrient agar

PCR and CHAS

Blotting grade non-fat dry milk powder blocker
Anti-DIG-POD (Roche Diagnostics, Cat. no. 11207733910)
BSA, non-acetylated bovine serum albumin, 20 mg/mL
DIG-11-dUTP, alkali stable (Roche Diagnostics, Cat. no. 11 558 706 910)
dNTP mix, PCR grade, 10 mM
Ethanol, 95%
Formamide
HotStarTaq (Qiagen, Cat. no. 203205). No substitutions permitted.
10% N-Lauroylsarcosine solution
Magnesium chloride (MgCl₂)
Sodium phosphate, monobasic, monohydrate (NaH₂PO₄·H₂O)
Sodium phosphate, dibasic, anhydrous (Na₂HPO₄)
Sodium citrate
Sodium chloride (NaCl)
20X SSC
10% Sodium dodecyl sulfate (SDS) solution
Tetramethylbenzidine – TMB Membrane peroxidase substrate, KPL brand (Mandel, Cat. no. 50-77-03). No substitutions permitted.
Triton X-100
Trizma base
Tween 20
DNase and RNase free water (Ultrapure or molecular grade)

Control cultures

Positive controls:

E. coli O26 containing vt1 and/or vt2 genes
E. coli O103 containing vt1 and/or vt2 genes
E. coli O111 containing vt1 and/or vt2 genes
E. coli O145 containing vt1 and/or vt2 genes
E. coli O157:H7 containing vt1 and vt2 and eae and hlyA genes (e.g., ATCC 35150)

Negative control:

Generic E. coli (negative for vt1, vt2, hlyA, and eae genes as well as the priority serogroup markers)

Preparation of media and reagents

Vancomycin + Cefsulodin stock solution (antimicrobial solution for mTSB) — for 10 mL of solution:

Vancomycin hydrochloride — 10 mg
Cefsulodin sodium salt hydrate — 3 mg
Dissolve the antimicrobials in ultrapure water (e.g. milliQ, nanopure) and sterilize the solution by passing through a 0.22 µm filter into a sterile container such as a Falcon tube. Store at 4 °C for up to 2 weeks. The antimicrobial solution is diluted 1 in 100 in the mTSB enrichment broth to give a final concentration of 10 µg/mL vancomycin and 3 µg/mL cefsulodin.

Rainbow Agar dehydrated media (500 mL):

80102 Rainbow Agar O157 30 g packet (approx. 25 plates)
Prepare according to manufacturer’s instructions. After pouring plates, ensure they are thoroughly dry before use or storage by leaving the plates open in a clean laminar flow hood for 15 minutes. Store in the dark at 4–10 °C for up to two weeks.

1% Tween 20:

Add 1 mL Tween 20 to 99 mL ultrapure water (e.g., milliQ, nanopure). Autoclave at 121 °C, 15 psi for 15 minutes. Store at room temperature for up to 1 year.

20X SSC — 500 mL (can be purchased ready-made from VWR, Product No. CA12001-388):

Sodium chloride — 87.66 g (3 M)
Sodium citrate — 44.1 g (0.3 M)
Adjust pH to 7.0 with 5 N HCl
Ultrapure water (e.g. milliQ, nanopure) — to 500 mL
Dissolve reagents in 400 mL ultrapure water. Adjust the pH to 7.0 with 5 N HCl, and then adjust the volume to 500 mL. Autoclave at 121 °C, 15 psi for 15 minutes. Store at room temperature for up to 1 year.

1 M Tris HCl pH 8.0 — 100 mL (can be purchased ready-made from Sigma, Product No. T2694):

Add 12.11 g of Trizma base to a beaker containing 80 mL ultrapure water (e.g. milliQ, nanopure). Stir until dissolved. Adjust pH to 8.0 with 5 N HCl. Add water to 100 mL and transfer to a flask suitable for autoclaving. Autoclave at 121 °C, 15 psi for 15 minutes. Store at room temperature for up to 1 year.

1 M Magnesium Chloride — 100 mL (can be bought ready-made from Sigma, Product No. M1028-100ML):

Add 20.33 g of magnesium chloride to a beaker containing 80 mL ultrapure water (e.g. milliQ, nanopure). Stir until dissolved. Add water to 100 mL and transfer to a flask suitable for autoclaving. Autoclave at 121 °C, 15 psi for 15 minutes. Store at room temperature for up to 1 year.

5 M Sodium Chloride — 100 mL:

Add 29.22 g of sodium chloride to a beaker containing 80 mL ultrapure water (e.g. milliQ, nanopure). Stir until dissolved. Add water to 100 mL and transfer to a flask suitable for autoclaving. Autoclave at 121 °C, 15 psi for 15 minutes. Store at room temperature for up to 1 year.

High Salt Buffer (pH 8.0) — 100 mL:

1 M Tris-HCl — 10 mL (0.1 M)
1 M Magnesium chloride (MgCl₂) — 1 mL (0.01 M)
5 M Sodium chloride — 3 mL (0.15 M)
Ultrapure water (e.g. milliQ, nanopure) — to 100 mL
To make the High Salt Buffer, mix the components in a sterile container. Store at room temperature for up to 1 year.

2× Hybridization solution (HS) — 200 mL:

20 × SSC — 100 mL (10 ×)
10% Sodium dodecyl sulfate (SDS) — 800 µL (0.04%)
10% N-lauroylsarcosine — 4.0 mL (0.2%)
Blocker (non-fat dry milk powder) — 4.0 g (2.0%)
Ultrapure water (e.g. milliQ, nanopure) — to 200 mL
These solutions should be filter sterilized (do not filter sterilize or autoclave the 2X hybridization solution). A 20% N-lauroylsarcosine solution is available from Sigma (L7414); if using, adjust volume to 2.0 mL.
In a beaker with a stir bar, add sodium dodecyl sulfate and N-lauroylsarcosine to 60 mL ultrapure water. Add 100 mL 20X SSC, mix (if detergents precipitate, heat in a 37 °C water bath). Add blocker and stir vigorously for at least 1 hour, until well dissolved. Let the solution stand until bubbles disappear and add water to 200 mL. Do not autoclave; store in 25 mL aliquots at -20 °C for up to 1 year.

1× HS + 50% formamide (HSF):

1 part 2× HS + 1 part formamide
This solution should be prepared fresh the day of the experiment.

PBST pH 7.0–7.4 — 1 L:

Sodium chloride (NaCl) — 8.5 g
Sodium phosphate monobasic, monohydrate (NaH₂PO₄·H₂O) — 0.28 g
Sodium phosphate dibasic, anhydrous (Na₂HPO₄) — 1.1 g
Tween 20 — 0.5 mL
Ultrapure water (e.g. milliQ, nanopure) — to 1 L
Add the first three reagents to 800 mL water, stir until completely dissolved. pH solution and, if necessary, adjust to 7.0–7.4 using hydrochloric acid or sodium hydroxide before adding Tween 20. Add the Tween 20 and then add water to 1 L. There is no need to autoclave this solution. It can be prepared in a large volume (4–8 L) and stored at room temperature for up to 1 year.

PBST-B:

PBST + 0.5% (w/v) non-fat dry milk powder blocker
Add 0.5 g blocker to 100 mL PBST. This can be prepared ahead of time, dispensed into tubes in 25 mL aliquots, and stored at -20 °C for up to 1 year.

PBST-B + anti-DIG-POD:

1 in 2000 dilution of Fab fragments of sheep anti-digoxigenin-conjugated with horse radish peroxidase (anti-DIG-POD) in PBST-B
1 µL anti-DIG-POD in 2 mL of PBST-B
This solution should be prepared the day of the experiment and protected from light until use.

1% Triton X-100 — 50 mL:

0.5 mL Triton X-100 in 49.5 mL ultrapure water (e.g. milliQ, nanopure). Filter sterilize. Store at room temperature for up to 1 year.

Oligonucleotide preparation and storage:

Dried oligonucleotides: resuspend dried oligos in 1X TE (Tris-EDTA buffer pH 8, 10 mM Tris, 1 mM EDTA) at a 100 µM concentration and store at -20 °C. To avoid repeated freeze/thaw cycles of oligos it is advised to prepare 25 µL aliquots of 100 µM stocks dispensed into screw-cap tubes — the same tubes used for probe dilution and denaturation.
“Lab-ready” oligonucleotides may be purchased at 100 µM concentration. A source used by one of the laboratories is Integrated DNA Technologies, Inc. Aliquot as described above.
Preparation of F+R primer pair mixes: to reduce the number of pipetting steps when preparing the PCR master mix, the forward and reverse primer pair for a given target may be mixed in equimolar amounts as follows: mix 25 µL of each the 100 µM forward and reverse primer stocks for a given primer pair (F+R). In this mix the primers are now at a concentration of 50 µM. Store the 50 µL aliquots of F+R mix at -20 °C. When preparing the PCR master mix, dilute the 50 µM aliquot of F+R primer mix 1 in 10 in 1X TE, to a working concentration of 5 µM (e.g. 5 µL F+R primer mix added to 45 µL 1X TE).
Aliquoting oligonucleotide probes: dispense oligonucleotide probe aliquots (10 or 20 µL are convenient volumes) in the same 1.5 mL screw-cap tubes used for the denaturation step.

PCR master mix preparation

VT-PCR master mix — master mix may be prepared in larger quantities, aliquoted in portions of >150 µL into screw-cap 1.5 mL tubes, and stored at -20 °C for up to 2 months:

| Reagent | Volume (µL) | Final |
| Molecular Grade Water | 32.25 | |
| 10 × HotStar Taq PCR Buffer | 5 | 1× |
| dNTP mix (10 mM) | 1 | 0.2 mM |
| BSA non-acetylated (20 mg/mL) | 1.25 | 0.5 µg/µL |
| VT1-F (10 µM) | 1 | 0.2 µM |
| VT1-R (10 µM) | 1 | 0.2 µM |
| VT2-F (10 µM) | 1 | 0.2 µM |
| VT2-R (10 µM) | 1 | 0.2 µM |
| IAC-1 (10 µM) | 0.5 | 0.1 µM |
| IAC-2 (10 µM) | 0.5 | 0.1 µM |
| Qiagen HotStar Taq (5 U/µL) | 0.5 | 2.5 U |
| Template (lysate) | 5 | |
| Total Volume | 50 | |

EHEC multiplex PCR master mix — master mix may be prepared in larger quantities, aliquoted in portions of >150 µL into screw-cap 1.5 mL tubes, and stored at -20 °C for up to 2 months:

| Reagent | Volume (µL) | Final |
| Molecular grade water | 13 | |
| 10 × HotStar Taq PCR Buffer | 8 | 1.6 × |
| dNTP mix (10 mM) | 1 | 0.2 mM |
| DIG-11-dUTP (1 nmole /µL) | 0.5 | 10.0 µM |
| wzxO26 F+R mix (5 µM each) | 3 | 0.3 µM |
| wzxO103 F+R mix (5 µM each) | 2 | 0.2 µM |
| wzxO111 F+R mix (5 µM each) | 2 | 0.2 µM |
| wzxO145 F+R mix (5 µM each) | 2 | 0.2 µM |
| O157 F+R mix (5 µM each) | 2 | 0.2 µM |
| HLYA F+R mix (5 µM each) | 3 | 0.3 µM |
| EAE F+R mix (5 µM each) | 3 | 0.3 µM |
| VT1 F+R mix (5 µM each) | 2 | 0.2 µM |
| VT2 F+R mix (5 µM each) | 2 | 0.2 µM |
| IAC 1+2 mix (5 µM each) | 1 | 0.1 µM |
| Qiagen HotStar Taq (5U/µL) | 0.5 | 2.5 U |
| Template | 5 | |
| Total Volume | 50 | |

Oligonucleotide sequences

Multiplex PCR primers (sequences 5′ → 3′):

Primer reference notes: wzx O26 PCR primers (283 bp amplicon, designed from NCBI sequence no. DQ196413); wzx O103 PCR primers (205 bp amplicon, designed from NCBI sequence no. AY532664); wzx O111 PCR primers (229 bp amplicon, designed from NCBI sequence no. AY863412); wzx O145 PCR primers (211 bp amplicon, designed from NCBI sequence no. AY863412); O157 PCR primers (259 bp amplicon from rfbE gene, Paton and Paton, 1998); VT1 PCR primers (182 bp amplicon from vt1 gene, designed from NCBI #M19473); VT2 PCR primers (200 bp amplicon from vt2 gene, designed from NCBI # X07865); eae PCR primers (384 bp amplicon from eae gene, Paton and Paton, 1998); hlyA PCR primers (534 bp amplicon from hlyA gene, Paton and Paton, 1998); IAC PCR primers (40 bp amplicon from Leggate & Blais, 2006).

Oligonucleotide probes (sequences 5′ → 3′):

Probe reference notes: wzx O26 Probe (50-mer, 39% G+C, designed from NCBI sequence no. DQ196413); wzx O103 Probe (50-mer, 47% G+C, designed from NCBI sequence no. AY532664); wzx O111 Probe (50-mer, 39% G+C, designed from NCBI sequence no. AY863412); wzx O145 Probe (50-mer, 57% G+C, designed from NCBI sequence no. AY863412); O157 Probe (50-mer, 48% G+C, designed from rfb gene sequence, Bilge et al., 1996); VT1 Probe (50-mer, 51% G+C, designed from NCBI #M19473); VT2 Probe (50-mer, 51% G+C, designed from NCBI # X07865); eae Probe (50-mer, 54% G+C, designed from NCBI sequence no. AF065628); hlyA Probe (50-mer, 46% G+C, designed from EHEC hlyA gene sequence Schmidt et al., 1995); IAC probe (40-mer, 45% G+C, Leggate & Blais, 2006).

0.1% (w/v) Peptone Water — 100 mL
Peptone — 0.1 g
Distilled water — to 100 mL
Nutrient Agar (plates or slants)
Basal Medium
Peptone — 5.0 g
Beef extract — 3.0 g
Agar — 15.0 g
Distilled water — 1.0 L
pH 6.8 ± 0.2
Complete Medium: suspend the ingredients in 1.0 L of distilled water and heat to boiling with frequent swirling; dispense into tubes or plates and autoclave at 121 °C for 15 min; cool the tubes in a slanted position
10% N-Lauroylsarcosine solution — 100 mL
N-Lauroylsarcosine — 10 g
Distilled water — to 100 mL
10% Sodium Dodecyl Sulfate (SDS) solution — 100 mL
Sodium dodecyl sulfate — 10 g
Distilled water — to 100 mL
5 N HCl — use as supplied (commercially available)

Sample preparation

Handling of sample units

In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated or frozen, depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.
Analyse sample units as soon as possible after their receipt in the laboratory.

Preparation for analysis

Have sterile diluent prepared, pre-warmed to 35 °C ± 1 °C before use.
Clean the surface of the working area with a suitable disinfectant.

Preparation of sample

To ensure a representative analytical unit, agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit.
Place a 25 g sample of the food (the analytical unit) into 225 mL of modified tryptone soya broth (mTSB) in a sterile filter-stomacher bag.
Stomach the sample for 2 minutes (the source uses “stomach” as a verb).

Procedure

Enrichment

Incubate the homogenized sample without shaking for 4 hours at 42 °C ± 0.5 °C
↓
Following the 4 hours of enrichment, add 2.5 mL of the vancomycin + cefsulodin solution (the antimicrobial solution contains 1 mg/mL vancomycin + 0.3 mg/mL cefsulodin) to the 225 mL of mTSB — this gives a final antimicrobial concentration in the enrichment broth of 10 µg/mL vancomycin and 3 µg/mL cefsulodin. Working in a biological safety cabinet, add the antimicrobial solution to the sample slowly (to avoid a high concentration of the antimicrobial solution on the food sample) and mix manually for 30 seconds by gently squeezing the bag
↓
Continue the enrichment by incubation overnight for 16–20 hours at 42 °C ± 0.5 °C without shaking
↓
Set up a positive and a negative control at the same time: use a vt1+, vt2+, hlyA+ and eae+ strain of E. coli O157:H7 as a positive control, and a generic E. coli (negative for vt1, vt2, hlyA, and eae genes as well as the priority serogroup markers) as a negative control

Screening of enrichment broths by VT-PCR

Prepare template DNA from the enrichment broths using InstaGene Matrix:
↓
To avoid working with a large volume of enrichment broth, use a sterile pipet to transfer a minimum of 5 mL of broth into a labelled test tube
↓
To extract DNA from the enrichment broth, transfer a 1 mL aliquot of the broth into a sterile 1.5 mL centrifuge tube. Store the remainder of the enrichment broths at room temperature, until the results of the screening VT-PCR are known
↓
Centrifuge the tube at 4 °C for 3 minutes at 10,000 x g
↓
Remove the supernatant from the tube and re-suspend the pellet in 1 mL of sterile, 1% (v/v) Tween 20 solution. Set up a lysis blank negative control by adding 1 mL of sterile, 1% (v/v) Tween 20 solution to a sterile 1.5 mL centrifuge tube and carrying it through the rest of the procedure. Vortex at high speed for 10 seconds
↓
Centrifuge the tube at 4 °C for 3 minutes at 10,000 x g
↓
Remove and discard the supernatant from the pellet. Mix InstaGene Matrix on a magnetic stirrer to maintain the matrix in suspension (the product bottle comes with a stir bar). Add 200 µL of InstaGene Matrix to the pellet (use a large bore pipet tip, such as a 1000 µL tip) and vortex for at least 10 seconds
↓
Transfer the tube to a heating block or water bath and incubate at 56 °C for 15 to 30 minutes
↓
Mix the contents by vortexing for 10 seconds and place the tube in a 100 °C heating block, or boiling water bath, for 8 minutes
↓
Cool the tube on ice for 5 minutes, vortex the tube for 10 seconds, then centrifuge at 4 °C for 3 minutes at 10,000 x g
↓
Use 5 µL of supernatant as template for a 50 µL PCR reaction
↓
Label reaction tubes. Prepare the VT-PCR master mix and dispense 45 µL into reaction tubes. Add 5 µL extracted DNA template to corresponding tubes (including positive and negative controls)
↓
Insert tubes in a thermal cycler and proceed with the PCR amplification using the VT-PCR cycling program below
↓
Analyse the PCR products by FlashGel electrophoresis or an equivalent procedure: load 5 µL of FlashGel 50 bp-1.5 kb DNA marker into the first well of a FlashGel 2.2% DNA cassette; mix 2 µL of PCR product with 2 µL of ultrapure or molecular grade water and 1 µL of 5× FlashGel loading dye; load the prepared samples into the remaining wells of the FlashGel DNA cassette
↓
Run FlashGel for approximately 6.5 minutes at 275 V. Under these conditions, both VT bands and the IAC should be resolved as follows: VT1, 182 bp; VT2, 200 bp; IAC, 40 bp
↓
Enrichment broths testing positive for the vt1 and/or vt2 genes are plated on Rainbow agar and screened for strain isolation. Samples positive for vt1 and/or vt2 are retained for strain isolation. Enrichment broths testing negative for both vt1 and vt2 genes and positive for IAC are discarded and the sample is considered negative. Failure of the IAC reaction is evidence of inhibition of the PCR by sample matrix contaminants, failure of one of the detector reagents, or omission of a critical component in the procedure — repeat the VT-PCR for samples with a failed IAC reaction

VT-PCR cycling program — the thermal cycler program should be programmed for the following sequence of cycling parameters:

Strain isolation

Plating of enrichment broths:
↓
Prepare serial dilutions of the enrichment broth to 10⁻⁶ by mixing 1 mL aliquots of the enrichment broth with 9 mL of 0.1% peptone water
↓
Spread-plate 100 µL of the 10⁻⁴, 10⁻⁵ and 10⁻⁶ dilutions onto Rainbow agar
↓
Incubate plates for 16–24 hours at 35 °C ± 1 °C. Rainbow agar plates should be incubated and stored in the dark

Note: If a suitable number of individual colonies are not observed on the Rainbow agar plates following plating of the 10⁻⁴, 10⁻⁵ and 10⁻⁶ dilutions of the VT positive enrichment broths, repeat the plating using a different dilution range (e.g., 10⁻¹, 10⁻² and 10⁻³) to generate plate dilutions with the largest number of individual colonies, ideally 50–100 colonies per plate. If fewer than 50–100 colonies are generated after plating the lowest dilution (least dilute), subject all colonies to the colony screening. If no colonies are observed after plating the enrichment broth, the sample is considered negative.

Colony screening:
↓
For VT positive enrichment broths, select and pool isolated colonies to screen by VT-PCR for vt1 and vt2. Select colonies at random, bearing in mind the highly variable characteristics of EHEC. It is especially important to pick colonies at random from the Rainbow agar, so that all the various phenotypes are included in the pool. For batch-wise colony screening, start with 2 pools of 10 colonies (pick colonies from the plate dilution(s) showing the largest number of individual colonies, ideally 50–100 colonies per plate). Pick a portion of a single colony with a loop, touch the loop onto a non-selective agar plate such as BHI or nutrient agar (labelled with a grid to keep track of individual colonies). Suspend the remainder of the colony in 200 µL 1% (v/v) Triton X-100 in a sterile 1.5 mL microfuge tube with a secured cap (e.g., screw-cap). Repeat the procedure, and suspend the remaining 9 colonies that constitute a pool into the same microfuge tube. The non-selective plate is incubated 18–24 hours at 35 °C ± 1 °C
↓
Prepare the template for PCR by vortexing the tubes containing the colony pool for 10 seconds, and then place the tubes into a 100 °C heating block for 10 minutes. Cool the resulting lysates for 5 minutes at room temperature and pulse-spin for a few seconds to bring down condensation accumulated under the tube cap. Use 5 µL of the supernatant as the PCR template
↓
Test each colony pool for the presence of verotoxin genes using the screening VT-PCR protocol above. In the absence of any VT-positive results from a colony pool, pick another two pools of 10 colonies each and process in like manner. Repeat up to three rounds if necessary, for a total of 60 colonies
↓
To identify VTEC from positive colony pools, go back to the isolated colonies from that pool (on the non-selective plate) and test them individually for verotoxin genes by VT-PCR. Lysates from these isolates may be stored at 4 °C for the remainder of the work day and used as template for the EHEC multiplex PCR
↓
Individual colonies on the non-selective plate which test positive for vt1 and/or vt2 are characterized by the EHEC multiplex PCR and CHAS procedure

Characterization of VTEC isolates by EHEC multiplex PCR and cloth-based hybridization array system (CHAS)

EHEC multiplex PCR:
↓
Label reaction tubes. Prepare the EHEC multiplex PCR master mix and dispense 45 µL into reaction tubes
↓
If template DNA is available from the colony screening above, proceed to the template addition. If template DNA is not available, pick a portion of the vt1 and/or vt2 positive isolates from the non-selective plate and suspend in 200 µL 1% (v/v) Triton X-100 held in a 1.5 mL microfuge tube with a secured lid. Set up a lysis buffer negative control tube by dispensing 200 µL 1% (v/v) Triton X-100 and carrying it through the rest of the procedure. Set up positive and negative controls with colonies from the strains used in the enrichment step. Mix contents by vortexing for 10 seconds, and place the tube in a 100 °C heating block for 10 minutes. Cool lysates for 5 minutes at room temperature, and pulse-spin for a few seconds to bring down condensation accumulated under the tube cap

Note: A mixture of laboratory strains presenting all of the molecular markers targeted by the assay can be used as a PCR and EHEC CHAS positive control for each sample set. A mixture of all five priority EHEC serogroups strains can prepared by combining one colony of each into 200 µL of 1% (v/v) Triton X-100 and then lysing as described above.
↓
Add 5 µL of the colony lysate supernatant to the corresponding PCR reaction tube
↓
Insert tubes in a thermal cycler and proceed with the PCR amplification using the EHEC multiplex PCR cycling program below

Note: The PCR may be run overnight and the amplicons stored at 4 °C for up to 24 hours. For longer storage, PCR tubes containing amplicons may be stored at -20 °C for up to one week.

EHEC multiplex PCR cycling program — the thermal cycler program should be programmed for the following sequence of cycling parameters:

EHEC CHAS — preparation of cloth strips:
↓
Use polyester cloth strips 3 x 6 cm (DuPont, Sontara 8100) bearing a printed sample location grid with 10 cells. Place the cloth strips onto the filtration apparatus (the method includes an illustration of the filtration apparatus setup). Several strips can be processed simultaneously, provided they do not overlap. Saturate the cloth strips with 95% ethanol for 1 minute
↓
Wash the strips 7 times with ultrapure or molecular grade water, using a squeeze bottle: stop the vacuum, saturate the strips with water, and open the vacuum to remove excess water
↓
Leaving the vacuum on, pick up the cloth strips with forceps and place in an empty petri dish or tray. Do not stack the cloth strips. Dry the strips overnight uncovered, at room temperature away from light. Once dry, strips may be stored stacked inside a clean dry container (e.g., Petri dish or plastic bag) at room temperature, away from light

Note: When a large number of strips are washed and placed to dry in a drawer or cupboard, the drying time required should be extended, as the increased humidity caused by evaporation will lengthen drying time. An indication that the strips are not completely dry is that when the probe solution is applied to the cloth, the wet spots are not visible on the cloth.

EHEC CHAS — application of probe to cloth strips (these steps can be performed while the EHEC multiplex PCR is running):
↓
Oligonucleotide probes for O26, O103, O111, O145, O157, eae, hlyA, VT1, VT2 and IAC are as described under Oligonucleotide sequences above. Probe stocks are prepared in 100 µM concentrations in TE buffer. Probes are diluted in High Salt Buffer containing 30% (v/v) ethanol to a final coating concentration of 10 µM as follows: to prepare 100 µL of probe coating solution, mix 10 µL of 100 µM oligo probe stock with 59 µL High Salt Buffer in a 1.5 mL screw-cap tube; denature the probes by placing in a heat block at 100 °C for 10 minutes; snap-chill the probes by immersion in ice water for 5 minutes; centrifuge tubes for a 10 second pulse to bring down condensate and return the tubes to the ice bucket immediately; add 31 µL of cold 95% ethanol to obtain a final probe concentration of 10 µM; mix well by flicking the tube with the fingers; keep tubes on ice during the procedure (or a chiller block at 4 °C)

Note: It is recommended that the probe coating solution is prepared and used in volumes of no larger than 200 µL at a time. When spotting a large number of cloth strips, these procedures should be performed in batches. Do not centrifuge the tubes after adding and mixing the ethanol, or the solution will separate into two phases.
↓
Place the cloth strips to be spotted in square Petri plates or another suitable tray (it must fit in the UV crosslinker) and use a pipettor to spot 7 µL of each of the ten denatured probes (O26, O103, O111, O145, O157, eae, hlyA, VT1, VT2 and IAC) in discrete spots onto each cloth strip (the method’s spotting pattern figure places O26/O103/O111/O145/O157 in cells 1–5 and VT1/VT2/eae/hlyA/IAC in cells 6–10; a multi-channel pipettor may be used to spot rows of five probes simultaneously)
↓
Dry the probe-coated strips uncovered in a 35–37 °C incubator for 30–40 minutes
↓
Covalently bind the probes to the cloth strip by exposing to UV light using a UV cross-linker set at 254 nm, 120 mJ/cm² (this step is critical to ensure that probes will be bound to the strips)
↓
Saturate each strip with 1.5 mL of 1× Hybridization Solution to block non-specific binding sites on the cloth. Block the cloth strips for at least one hour at 35–37 °C with the lids on (the strips can be blocked for up to 3 hours)
↓
Use forceps to transfer the cloth strips to the filtration apparatus for washing. Several strips can be processed simultaneously, provided they do not overlap
↓
Wash the strips 5 times by saturating the strips on the filter with PBST using a squeeze bottle and applying vacuum suction between washes
↓
While the vacuum is still on, use forceps to pick up the cloth strips and place them in Petri plates. Strips can be used immediately, or placed in a single layer in an uncovered square Petri plate (or other appropriate container) away from light to air-dry at room temperature overnight
↓
Dried, probe-coated strips can be stored stacked in square Petri plates, sealed with Parafilm at 4 °C for up to two weeks. Alternatively, dried probe-coated strips can be sealed in vacuum pouches and stored at 4 °C for up to 6 months

EHEC CHAS — hybridization and colorimetric detection:
↓
Manipulate cloth strips using tweezers; rinse tweezers in a small beaker containing water or PBST buffer between each treatment
↓
Place probe-coated strips in individual Petri plates (or use divided Petri plates to assay two strips per plate). A pencil can be used to label strips (e.g., with dots corresponding to the PCR reaction or sample number)
↓
Transfer 40 µL of each DIG-labelled multiplex PCR product into a 1.5 mL microcentrifuge tube (use lid-locks or screw-cap 1.5 mL tubes to prevent the tubes from opening while heating). Denature PCR products in a heating block at 100 °C for 10 minutes, followed by rapid chilling on ice water for 5 minutes. Pulse spin tubes in a centrifuge to bring down condensation before opening tubes during the next step

Note: The remaining 10 µL of PCR reaction is stored at -20 °C until results have been obtained, in the event the PCR products have to be visualized using agarose gel electrophoresis.
↓
Add 1.5 mL of ice-cold hybridization solution containing 50% formamide (HSF) to each 1.5 mL microcentrifuge tube containing 40 µL of denatured, labelled PCR products. Vortex for 2 to 3 seconds to mix solution. Pulse spin in a microcentrifuge before opening
↓
Carefully saturate each cloth strip with the denatured PCR product in HSF and incubate for 15 minutes at 45 °C ± 1 °C with the cover on
↓
Transfer the strips to the filtration apparatus using forceps. Wash the strips 5 times with PBST (using a squeeze bottle) and applying vacuum suction between washes
↓
Keeping the vacuum open, transfer the strips to a dry, clean Petri dish or tray. Several strips can be placed in the same Petri dish or tray without overlapping
↓
Add 1.5 mL of a 1 in 2000 dilution of anti-DIG-POD in PBST-B to each strip. Incubate for 10 minutes at room temperature. Wash the strips 5 times with PBST. More than one strip can be washed at a time for this step. Use forceps to transfer washed strips to a square Petri dish

Note: The anti-DIG-POD in PBST-B solution must be prepared fresh the day of the experiment. During the incubation, dispense the volume of tetramethylbenzidine (TMB) membrane peroxidase substrate that will be used for the experiment into a polypropylene tube and keep at room temperature away from the light to warm up.
↓
Add 1.5 mL of TMB to each strip. Cover with aluminium foil and allow the colour to develop for up to 10 minutes. Blue/purple spots coinciding with an appropriate probe indicate a positive reaction
↓
Using forceps, pick up strips and dip in distilled water to rinse. Place on paper towels to blot excess moisture. Record results and, if desired, photograph the strips using a camera or a UV transilluminator on the same setting used for gels stained with ethidium bromide

Interpretation of CHAS results

Formation of a blue precipitate on the CHAS strip coinciding with a given probe is considered a positive reaction for the gene sequence corresponding to that probe.
Isolates are considered priority EHEC when colour reactions occur with the eae, one of the O antigen markers and/or hlyA, in addition to either of the verotoxin (vt1 and/or vt2) gene probes on the array.
The negative control (generic E. coli) should only produce a colour reaction with the IAC probe feature.
The E. coli O157:H7 positive control should produce a colour reaction with the O157, vt1, vt2, eae, hlyA and IAC probe features.
The positive control mixture of template DNA from all target strains (if used) should produce a colour reaction with all probe features in the array.
For samples producing reactions with one or more VTEC features, the IAC signal may be weak due to consumption of PCR reagents. In this case, the reactivity observed at the other VTEC features is taken as confirmation of the validity of the test procedure.
Negative test results are not considered valid if a colour reaction occurs with the IAC probe on the cloth strip. Failure to see an IAC reaction is evidence of either (i) inhibition of the PCR by sample matrix contaminants; (ii) failure of one of the detector reagents; or (iii) omission of a critical component in the procedure (e.g., PCR reaction mixture). Repeat the CHAS procedure for samples with a failed IAC reaction.

Further characterization of colonies by O-Typer (optional)

In the event that testing is targeting a VTEC strain which does not fall within the group of priority serogroups designated herein (e.g., in an outbreak investigation), the O antigen may be verified by subjecting isolates to the O Typer (Gilmour, et al. 2007).

Precautions

The laboratory Supervisor must ensure that completion of the analysis described in this method is done in accordance with the International Standards reference “ISO/IEC 17025:2005 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories”.
It is the responsibility of the laboratory to ensure equivalency if any variations of the media formulations listed here are used (either product that is commercially available or made from scratch).
It is the responsibility of each laboratory to ensure that the temperatures of the incubators or water baths are maintained at the recommended temperatures. Where 35 °C is recommended in text of the method, the incubator may be at 35 °C ± 1.0 °C. Similarly, lower temperatures of 30 °C or 25 °C may be ± 1.0 °C. However, where higher temperatures are recommended, such as 43 °C or 45.5 °C, it is imperative that the incubators or water baths be maintained within 0.5 °C due to potential lethality of the higher temperatures on the microorganism(s) being isolated.

References

Health Canada, Health Products and Food Branch. Isolation and identification of priority verotoxigenic Escherichia coli (VTEC) in foods. Compendium of Analytical Methods, Volume 3, Laboratory Procedure MFLP-52, November 2014.
Bilge, S.S., Vary, Jr., J.C., Dowell, S.F., and Tarr, P.I. 1996. Role of the Escherichia coli O157:H7 O side chain adherence and analysis of an rfb locus. Infection and Immunity, 64: 4795-4801.
Blais, B.W., and Martinez-Perez, A. 2011. A simple PCR-based macroarray system for detection of multiple gene markers in the identification of priority enterohemorrhagic Escherichia coli. Journal of Food Protection 74:365-372.
Gill, A., Martinez-Perez, A., McIllwham, S., and Blais, B. 2012. Development of a method for the detection of verotoxin-producing Escherichia coli in food. Journal of Food Protection 75: 827-837.
Gilmour, M.W., Olson, A.B., Andrysiak, A.K., Ng, L.K., and Chui, L. 2007. Sequence-based typing of genetic targets encoded outside of the O-antigen gene cluster is indicative of Shiga toxin-producing Escherichia coli serogroup lineages. Journal of Medical Microbiology. 56: 620-628.
Leggate, J, and Blais, B.W. 2006. An internal amplification control system based on primer-dimer formation for PCR product detection by DNA hybridization. Journal of Food Protection, 69:2280-2284.
Paton, A.W., and Paton, J.C. 1998. Detection and characterization of shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157. Journal of Clinical Microbiology, 36:598-602.
Schmidt, H., Beutin, L., and Karch, H. 1995. Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infection and Immunity, 63:1055-1061.

Access the GIFSQ Portal

Student Portal

Instructor Portal

Company Portal