SOP: Characterization of Verotoxigenic Escherichia coli O157:H7 Colonies by Polymerase Chain Reaction (PCR) and Cloth-Based Hybridization Array System (CHAS)

GIFSQ — Global Institute for Food Safety & Quality

Principle

The cloth-based hybridization array system (CHAS) is a DNA amplification and hybridization platform that allows colonies isolated on plating media to be checked for marker gene sequences associated with specific pathogenic bacteria. The E. coli O157:H7 CHAS picks up definitive gene sequences associated with this pathogen, giving sensitive and specific detection of multiple E. coli O157:H7 gene markers in a single assay. The key indicator genes rfbO157, fliCH7, vt1 and vt2 (encoding the O and flagellar antigenic determinants and verotoxin genes 1 and 2, respectively) are amplified in a multiplex PCR incorporating digoxigenin-11-dUTP; the amplicons are hybridized to an array of specific oligonucleotide probes immobilized on polyester cloth, and the bound digoxigenin label is detected by immunoenzymatic assay. An internal amplification control (IAC) shows whether PCR inhibition has occurred: it uses a primer pair with complementary 3′ ends, and self-hybridization of this primer pair generates a unique primer-dimer detected by a specific capture probe immobilized on polyester cloth.

This method is applicable to the identification of verotoxigenic Escherichia coli O157:H7 colonies isolated from foods by standard culture for verotoxigenic E. coli on validated plating media (see Sample preparation). It can be used to determine compliance with Sections 4 and 7 of the Food and Drugs Act and/or other relevant federal regulations. This revised method replaces MFLP-22, dated December 2019.

Apparatus

Equipment

• Biohazard hood and/or PCR workstation with UV lights
• Chiller block for 0.2 mL or 0.5 mL PCR tubes
• Filtration apparatus (VWR 70690-276) with vacuum (the method includes a set-up illustration for this apparatus) or verified equivalent
• Freezer capable of maintaining −20 °C
• Heat block capable of accommodating 1.5 mL microcentrifuge tubes and maintaining a temperature of 100 °C
• Incubators capable of maintaining 35 °C and 45 °C
• Microcentrifuge, capable of spinning 1.5 mL tubes at 12,000 × g
• Microcentrifuge, small (“pop centrifuge”)
• Micropipettors, adjustable to cover range of volumes from 1.0 to 1000 µL
• Photodocumentation system (Optional)
• Refrigerator capable of maintaining refrigeration temperature
• Thermal cycler equipped with a heated lid
• UV cross-linker that can be adjusted to specified settings
• Vortex mixer

Materials

• Polypropylene tubes, 50 mL
• Gloves, powder-free, disposable
• Ice and bucket (or equivalent)
• Screw-cap and regular microtubes, 1.5 mL, nuclease-free, sterile
• Microtube racks
• PCR tubes (0.2 mL or 0.5 mL), nuclease-free, sterile
• Segmented sterile Petri dishes (optional; may be used if assaying 2 strips/plate as described under Hybridization and colorimetric detection)
• Pipettes, 10 mL
• Micropipettor tips, filtered, nuclease-free, sterile
• Pre-cut polyester cloth (Sontara 8100) strips bearing a printed sample location grid with 5 cells, obtainable from the lab of Dr. Blais or purchased directly from Apace Screen Printing & Enterprises Ltd.; no substitutions permitted
• Racks, cold, to accommodate 0.2 mL, 0.5 mL, and 1.5 mL tubes
• Forceps
• Wash bottles
• Aluminium foil

Control cultures

• E. coli O157:H7 containing vt1 and vt2 genes (e.g., ATCC 35150)

Reagents

• Anti-DIG-POD (Roche Diagnostics 11207733910)
• Molecular grade non-fat dry milk powder blocker
• BSA, non-acetylated bovine serum albumin, 20 mg/mL
• DIG-11-dUTP, alkali stable (Roche Diagnostics 11558706910)
• dNTP mix, PCR grade, 10 mM
• Ethanol, 95%
• Formamide
• HotStarTaq (Qiagen 203205); no substitutions permitted
• Magnesium chloride (MgCl₂), potassium chloride (KCl), sodium chloride (NaCl), sodium phosphate monobasic (monohydrate) (NaH₂PO₄·H₂O), sodium phosphate dibasic (anhydrous) (Na₂HPO₄), sodium citrate, sodium dodecyl sulfate (SDS), N-Lauroylsarcosine
• Oligonucleotide probes, O157, H7, VT1, VT2, and IAC probes (see Oligonucleotide sequences)
• Primers, forward and reverse, O157, H7, VT1, VT2, and IAC (see Oligonucleotide sequences)
• 20X SSC
• Tetramethylbenzidine – TMB Membrane Peroxidase Substrate (1 component), KPL brand (Mandel Scientific 50-77-03); no substitutions permitted
• 100X Tris-EDTA buffer (pH 8.0)
• Triton X-100
• Trizma base
• Tween 20
• DNase and RNase free water (ultra-pure or molecular grade)

Oligonucleotide sequences

Multiplex PCR primers

(The primers print “FLICH7-F” and “FLICH7-R”; the gene is fliCH7.)

Oligonucleotide probes

Primer notes: O157 primers — 259 bp amplicon from rfbE gene (Paton and Paton, 1998); H7 primers — 625 bp amplicon from fliC gene (Gannon et al., 1997); VT1 primers — 182 bp amplicon from vt1 gene (Gauthier and Blais, 2005); VT2 primers — 200 bp amplicon from vt2 gene (Gauthier and Blais, 2005); IAC primers — 40 bp amplicon (Leggate and Blais, 2006). Probe notes: O157 probe — designed for this study from rfb gene sequence published by Bilge et al., 1996; H7 probe — designed for this study from fliC gene sequence published by Gannon et al., 1997; VT1 probe — designed for this study from published sequence GenBank accession M19473; VT2 probe — designed for this study from published sequence GenBank accession X07865; IAC probe (Leggate and Blais, 2006).

Preparation of reagents

1 M Tris HCl (pH 8.0) — 100 mL

Add 12.11 g of Trizma base to a beaker with 80 mL ultrapure water. Stir until dissolved. Adjust pH to 8.0 with 5 N HCl. Add water to 100 mL. Transfer to autoclave safe bottle. Autoclave at 121 °C, 15 psi for 15 min. Store at room temperature for up to 1 year.

1 M Magnesium Chloride — 100 mL

Add 20.33 g of magnesium chloride to a beaker with 80 mL ultrapure water. Add water to 100 mL. Stir until dissolved. Transfer to autoclave safe bottle. Autoclave at 121 °C, 15 psi for 15 min. 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 with 80 mL ultrapure water). Add water to 100 mL. Stir until dissolved. Transfer to autoclave safe bottle. Autoclave at 121 °C, 15 psi for 15 min. Store at room temperature for up to 1 year.

High Salt Buffer (pH 8.0) — 100 mL

• 10 mL of 1 M Tris-HCl (pH 8.0) — final concentration 0.1 M
• 1 mL of 1 M Magnesium chloride (MgCl₂) — final concentration 0.01 M
• 3 mL of 5 M Sodium chloride — final concentration 0.15 M
• Ultrapure Water (e.g. MilliQ, Nanopure) — to 100 mL

Mix the above stock solutions in a sterile container. Store at room temperature for up to 1 year.

2X Hybridization Solution (HS) Stock — 200 mL

• 100 mL of 20X SSC — final concentration 10X
• 800 µL of 10% Sodium dodecyl sulfate (SDS) — final concentration 0.04%
• The 10% SDS stock solution is used as supplied; filter sterilize before use.
• 2.0 mL of 20% N-lauroylsarcosine — final concentration 0.2%
• The 20% N-lauroylsarcosine stock solution is used as supplied; filter sterilize before use.
• 4.0 g of blotting grade non-fat dry milk powder blocker — final concentration 2.0%
• Ultrapure Water (e.g. MilliQ, Nanopure) — to 200 mL

(The 10% SDS and 20% N-lauroylsarcosine solutions should be filter sterilized.) Do not filter sterilize or autoclave the 2X hybridization solution. In a beaker with a stir bar, add 10% SDS and 20% 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 completely dissolved. Let the solution stand until bubbles disappear and add water to a final volume of 200 mL. Do not autoclave; store in 25 mL aliquots at −20 °C for up to 1 year.

1X HS + 50% Formamide

1 part 2X HS + 1 part formamide

PBST (pH 7.0–7.4) — 1 L

• 8.5 g of Sodium chloride (NaCl)
• 0.28 g of Sodium phosphate monobasic, monohydrate (NaH₂PO₄·H₂O)
• 1.1 g of Sodium phosphate dibasic, anhydrous (Na₂HPO₄)
• 0.5 mL of Tween 20
• Ultrapure water (e.g. MilliQ, Nanopure) — to 1 L

Add the first three reagents to 800 mL water, stir until completely dissolved. Determine the pH of the solution and, if necessary, adjust to 7.0–7.4 using hydrochloric acid or sodium hydroxide before adding Tween 20. Add Tween 20 and then add water to 1 litre. 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 in a carboy fit with a spigot for ease of use.

PBST-B

PBST + 0.5% (w/v) blotting grade non-fat dry milk powder blocker. Add 0.5 g blotting grade non-fat dry milk powder blocker to 100 mL PBST. This can be prepared ahead of time, dispensed into 50 mL polypropylene tubes in 25 mL aliquots, and stored at −20 °C for up 1 year. Do not autoclave.

PBST-B + Anti-DIG-POD

1:2000 dilution of Fab fragments of sheep anti-digoxigenin-conjugated with horseradish 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.

20X SSC — 500 mL

• 87.66 g of Sodium chloride — final concentration 3 M
• 44.1 g of Sodium citrate — final concentration 0.3 M
• 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 the volume to 500 mL. Autoclave at 121 °C, 15 psi for 15 min. Store at room temperature for up to 1 year.

1% Triton X-100 — 50 mL

0.5 mL Triton X-100 in 49.5 mL ultrapure water. Filter sterilize. Store at room temperature for up to 1 year.

PCR master mix preparation and cycling program

Master mix may be prepared in larger quantities, aliquoted in volumes of >150 µL into screw-cap 1.5 mL tubes, and stored at −20 °C for up to 2 months.

Master mix (50 µL reaction)

Final volume: 50.0 µL

PCR cycling program

Program the thermal cycler for the following sequence of cycling parameters:

Sample preparation

• Handle food samples and subject them to enrichment according to the applicable method in the Compendium of Analytical Methods for E. coli O157:H7, including plating on one of the validated media listed below.
• Pick an isolated colony with morphological characteristics typical of E. coli O157:H7 from one of the following media: Sorbitol MacConkey agar (SMAC), MacConkey agar (MAC), Modified Sorbitol MacConkey agar with Tellurite, Cefixime, and Cefsulodin (TCCSMAC), Modified Sorbitol MacConkey agar with Cefixime and Tellurite (CT-SMAC), Modified Hemorrhagic Coli Agar (mHC), CHROMagar, or RAPID’E. coli O157:H7 agar. An isolated colony from Nutrient Agar (NA) may also be characterized by this method if the colony was purified from one of the selective agars listed above, and/or had previously been confirmed to be E. coli.
• Suspend the colony in 200 µL of 1% (v/v) Triton X-100 in a 1.5 mL screw-cap centrifuge tube. For an uninoculated control, leave one tube of 1% Triton X-100 devoid of cells. Vortex to mix.
• Heat the tube at 100 °C for 10 minutes in a heat block. Cool in a rack on the bench to room temperature for 10 minutes. Spin 5 minutes at 12,000 × g in a microcentrifuge to pellet cell debris.

Procedure

Multiplex PCR

Aliquot 45 µL of PCR master mix into 0.2 or 0.5 mL PCR tubes for all samples, including one control reaction of PCR master mix and 5 µL water but no template, and one control reaction of PCR master mix with 5 µL 1% Triton X-100 devoid of template
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For each sample (but not the controls), add 5 µL of the prepared template (cell lysates) to the tubes containing PCR reaction mix
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Insert the tubes in a thermal cycler and amplify using the PCR cycling program given above
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If necessary, store the PCR products at 4 °C for up to 24 hours or at −20 °C for up to 3 months

Cloth strip preparation

Use 5-cell pre-printed cloth strips or cut the pre-printed polyester cloth into strips 2 cm × 4 cm
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Place the cloth strips onto the filtration apparatus (several strips can be processed simultaneously, provided they do not overlap); saturate the cloth strips with 95% ethanol for 1 minute
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Wash the strips 7 times with molecular grade water, using a squeeze bottle: to wash, stop the vacuum, saturate the strips with water, and open the vacuum to remove excess water
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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)
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Dry the strips uncovered overnight at room temperature away from light; once dry, store stacked flat inside a clean dry container (e.g. Petri dish or plastic bag) at room temperature, away from light

Probe preparation and strip coating

Dilute the probes to a stock solution of 100 µM (to prevent multiple freeze/thaw cycles of probe stocks, 10 µL aliquots of probes may be dispensed into 1.5 mL screw-cap tubes and diluted to coating concentration the day of the experiment)
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To prepare 100 µL of probe coating solution, place 10 µL of 100 µM oligo probe stock and 59 µL High Salt Buffer in a 1.5 mL screw-cap tube (the probes are diluted in High Salt Buffer containing 30% (v/v) ethanol to a final coating concentration of 10 µM)
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Denature the probes by incubating the tubes in a heating block at 100 °C for 10 minutes, followed by rapid chilling on ice for about 5 minutes; pulse spin in a centrifuge to bring down condensation
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Immediately add 31 µL of cold 95% ethanol to obtain a final probe concentration of 10 µM; mix well by flicking the tube with your fingers (do not vortex); keep the tubes on ice or a chiller block (do not centrifuge after mixing in the ethanol, or the solution will separate into two phases)
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Place the cloth strips to be spotted in a Petri plate, and use a micropipettor to spot 7 µL of each of the five denatured probes (O157, H7, VT1, VT2, and IAC), 1 cm apart on the cloth strips (a multipipettor may be used to spot all five probes simultaneously; set up the 0.2 mL PCR tubes (or tube strip) on a chiller block and, after mixing in the ethanol, transfer the chilled probe solutions into the tubes (or tube strip) in the proper order for spotting)
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Dry the probe-coated strips uncovered in a 35–37 °C incubator for 30 minutes
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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)
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Saturate each strip with 1.5 mL of 1X 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)
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Use forceps to transfer the cloth strips to the filtration apparatus for washing (several strips can be processed simultaneously, provided they do not overlap)
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Wash the strips 5 times by saturating the strips on the filter with PBST using a squeeze bottle and applying vacuum suction between washes
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While the vacuum is still on, use forceps to pick up the cloth strips and place them in Petri dishes (strips can be used immediately, or placed in a single layer in an uncovered Petri dish or other appropriate container away from light to air-dry at room temperature overnight)
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Store dried probe-coated cloth strips in a sealed, air-tight container, refrigerated in the dark for up to three months

Hybridization and colorimetric detection

Place probe-coated strips in individual Petri dishes; label each strip with a black marker or pencil spots in a way to reference the sample number (segmented Petri dishes can be used to assay two strips per dish)
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Transfer 40 µL of each DIG-labelled PCR product into a 1.5 mL screw-cap microcentrifuge tube; denature by incubating the tubes in a heating block for 10 minutes at 100 °C, followed by rapid chilling on ice for about 5 minutes; pulse spin in a microcentrifuge to bring down condensation (the remaining 10 µL of PCR reaction can be kept so that the PCR products can be visualized using agarose gel electrophoresis if required for troubleshooting reasons)
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Add 960 µL of ice-cold 1X Hybridization Solution containing 50% formamide (HSF) to each 1.5 mL centrifuge tube; vortex for 2 to 3 seconds to mix; pulse spin in a microcentrifuge before opening; continue to work on ice
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Carefully saturate each cloth strip, one per Petri dish (or two per segmented Petri dish), with the denatured PCR product in HSF; incubate the saturated strips in a 45 °C incubator for 15 minutes with lids on (the anti-DIG-POD in PBST-B solution must be prepared fresh, on the day of the experiment, either prior to starting the procedure or during this 45 °C incubation)
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Transfer the strips to the filtration apparatus (four strips may be washed at a time) using forceps; wash the strips 5 times by saturating the strips on the filter with PBST (using a squeeze bottle) and applying vacuum suction between washes (rinse forceps in a small beaker containing distilled water between each treatment)
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Keeping the vacuum open, transfer the strips to a dry Petri dish or tray (several strips can be placed in the same Petri dish or tray without overlapping)
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Add 1 mL of a 1:2000 dilution of anti-DIG-POD in PBST-B to each strip; incubate for 10 minutes at room temperature and protect from light (during this incubation, dispense the volume of tetramethylbenzidine (TMB) membrane peroxidase substrate that will be used for the experiment into a polypropylene tube; keep the tube at room temperature and protected from light)
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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 the lid of the Petri dish
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Add 1 mL of TMB to each strip; protect the strips from light and allow the colour to develop for up to 10 minutes (blue/purple spots coinciding with an appropriate probe indicate a positive reaction)
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Using forceps, pick up the 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 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 confirmed as verotoxigenic E. coli O157:H7 when colour reactions occur with the O157 and H7 probes, in addition to either of the verotoxin gene probes on the array (i.e. VT1 and/or VT2).
• Samples negative for O157, H7, VT1, and VT2 will only have a colour reaction with the IAC probe.
• Negative test results are considered valid if a colour reaction occurs with the IAC probe on the cloth strip. Failure to see an 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.

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 (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).
• No substitutions are permitted for the pre-cut polyester cloth strips, the HotStarTaq polymerase, or the TMB membrane peroxidase substrate.
• The UV cross-linking step is critical to ensure that probes will be bound to the strips.
• Do not filter sterilize or autoclave the 2X hybridization solution.
• The PBST-B + anti-DIG-POD solution must be prepared fresh on the day of the experiment and protected from light until use.
• Protect the TMB substrate from light.

References

• Health Canada. Characterization of Verotoxigenic Escherichia coli O157:H7 Colonies by Polymerase Chain Reaction (PCR) and Cloth-Based Hybridization Array System (CHAS). Compendium of Analytical Methods, Volume 3, Laboratory Procedure MFLP-22, February 2021. Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada.
• Bilge, S.S., Vary, J.C., Dowell, S.F. and Tarr, P.I., 1996. Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus. Infect. Immun., 64:4795-4801.
• Gannon, V., D’Souza, S., Graham, T., King, R.K., Rahn, K., and Read, S., 1997. Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains. J. Clin. Microbiol., 35:656-662.
• Gauthier, M. and Blais, B.W., 2005. Cloth-based hybridization array system for detection of toxin genes associated with major foodborne pathogenic bacteria. J. Rap. Meth. Auto. Microbiol., 13:243-256.
• Gilmour, M.W., Tracz, D.M., Andrysiak, A.K., Clark, C.G., Tyson, S., Severini, A. and Ng, L.K., 2006. Use of the espZ gene encoded in the locus of enterocyte effacement for molecular typing of shiga toxin-producing Escherichia coli. J. Clin. Microbiol., 44:449-458.
• Leggate, J. and Blais, B.W., 2006. An internal amplification control system based on primer-dimer formation for PCR product detection by DNA hybridization. J. Food Prot., 69:2280-2284.
• Nagano, I., Kunishima, M., Itoh, Y., Wu, Z., and Takahashi, Y., 1998. Detection of verotoxin-producing Escherichia coli O157:H7 by multiplex polymerase chain reaction. Microbiol. Immunol., 42:371-376.
• Ochoa, T.J., and Cleary, T.G., 2003. Epidemiology and spectrum of disease of Escherichia coli O157. Curr. Opin. Infect. Dis., 6:259-263.
• Osek, J., 2001. Multiplex polymerase chain reaction assay for identification of enterotoxigenic Escherichia coli strains. J. Vet. Diag. Invest., 13:308-311.
• Paton, A.W. and Paton, J.C., 1998. Detection and characterization of shiga toxigenic Escherichia coli by multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157. J. Clin. Microbiol., 36:598-602.
• Persson, S., Olsen, K.E.P., Ethelberg, S., and Scheutz, F., 2007. Subtyping method for Escherichia coli shiga toxin (verocytotoxin) 2 variants and correlations to clinical manifestations. J. Clin. Microbiol., 45:2020-2024.
• Health Canada. MFHPB-10: Isolation of Escherichia coli O157:H7/NM from foods and environmental surface samples. In Volume 2: Compendium of Analytical Methods. (GIFSQ SOP: Isolation of Escherichia coli O157:H7/NM from Foods and Environmental Surface Samples)