SOP: Determination of Verotoxin genes in Verotoxigenic Escherichia coli isolates

GIFSQ — Global Institute for Food Safety & Quality

Principle

This method determines the presence of the verotoxin genes in verotoxigenic Escherichia coli (VTEC) colonies isolated from foods. It can be used to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act and/or other relevant federal regulations.

Following the detection and isolation procedures, presumptive VTEC colonies from agars are subjected to a simple polymerase chain reaction (PCR) targeting the verotoxin 1 and/or 2 genes (VT1 and/or VT2), followed by analysis of the amplicons by rapid agarose gel electrophoresis (VT-PCR). The system includes an internal amplification control (IAC) to determine whether PCR inhibition has occurred, based on the incorporation of a template-independent primer pair with complementary 3′ ends. Individual colonies that test positive by VT-PCR are confirmed to possess the genes encoding the virulence factors verotoxin 1 and/or 2.

Apparatus

Equipment

• Biological safety cabinet and/or PCR workstation
• Chiller block for 0.2 mL or 0.5 mL PCR tubes
• Freezer capable of maintaining −20 °C
• Heat block capable of accommodating 1.5 mL-capacity microcentrifuge tubes and maintaining a temperature of 100 °C
• Microcentrifuge capable of spinning 1.5 mL tubes
• Micropipettors adjustable to cover the range of volumes from 1.0 to 1000 µL
• Photodocumentation system (optional)
• Refrigerator capable of maintaining 4 °C
• Thermal cycler equipped with a heated lid
• Vortex mixer
• FlashGel DNA System (Lonza Group Ltd.), or equivalent

Materials

• Gloves, disposable
• Screw-cap and regular microtubes, 1.5 mL-capacity, nuclease-free, sterile
• Microtube racks
• PCR tubes (0.2 mL or 0.5 mL), nuclease-free, sterile
• Pipettor tips, filtered, nuclease-free, sterile
• Cold racks, to accommodate 0.2 mL, 0.5 mL, and 1.5 mL tubes
• FlashGel DNA Cassette (2.2% agarose)

Reagents

• Rainbow Agar dehydrated media (Biolog, Inc., Cat. no. 80102)
• Brain Heart Infusion (BHI) agar or Nutrient Agar (NA)
• Bovine serum albumin (BSA), non-acetylated, 20 mg/mL
• dNTP mix, PCR grade, 10 mM
• HotStarTaq (Qiagen, Cat. no. 203205). No substitutions permitted
• Primers, forward and reverse, VT1, VT2, and IAC (sequences under Oligonucleotide sequences)
• DNase and RNase free water (Ultrapure or molecular grade)
• Triton X-100
• FlashGel Loading Dye (the source prints “FlashGel Loading Dye”; the procedure prints “5X Flash Gel” with a registered-trademark mark)
• FlashGel DNA Marker 50bp-1.5kb (the source prints “FlashGel DNA Marker 50bp-1.5kb”; the procedure prints “FlashGel 50 bp-1.5 kb DNA marker” with a registered-trademark mark)

Control cultures

• Positive control: E. coli O157:H7 containing vt1 and vt2 (e.g., ATCC 35150)
• Negative control: generic E. coli (negative for both verotoxin genes)

VT-PCR master mix

Master mix may be prepared in larger quantities, aliquoted in portions of > 150 µL into screw-cap 1.5 mL-capacity 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 (10mM) | 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 | |

Note: The 10 × HotStar Taq PCR Buffer is included with the Qiagen HotStarTaq polymerase kit. See Oligonucleotide sequences for the primer sequences.

VT-PCR cycling program

The thermal cycler program should be programmed for the following sequence of cycling parameters:

| Step | Temperature | Time |
| 1. HotStarTaq polymerase activation | 94 °C | 15 min |
| 2. 35 cycles of: Denaturation | 94 °C | 30 sec |
| 2. 35 cycles of: Annealing | 55 °C | 30 sec |
| 2. 35 cycles of: Extension | 72 °C | 1 min |
| 3. Final elongation | 72 °C | 2 min |
| 4. Hold | 4 °C | indefinitely |

Oligonucleotide sequences

| Primer name | Sequence (5′ → 3′) | Amplicon size (bp) |
| VT1-F | GTG GCA AGA GCG ATG TTA CGG TTT G | 182 |
| VT1-R | ATG ATA GTC AGG CAG GAC GCT ACT C | |
| VT2-F | ACG AGG GCT TGA TGT CTA TCA GGC G | 200 |
| VT2-R | GCG ACA CGT TGC AGA GTG GTA TAA C | |
| IAC-1 | CAT AAT ATC ACT CGC GTC CGT TGA AGC TTA | 40 |
| IAC-2 | GAC GAA ATC GTA AGC TTC AA | |

Notes: VT1 PCR primers — 182 bp amplicon from the vt1 gene, designed from NCBI #M19473. VT2 PCR primers — 200 bp amplicon from the vt2 gene, designed from NCBI #X07865. IAC PCR primers — 40 bp amplicon from Leggate & Blais, 2006.

Media formulations

• Nutrient Agar (NA)
• 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

Sample preparation

• Handle food samples and subject them to enrichment according to the applicable method in the Compendium of Analytical Methods for verotoxigenic E. coli, which includes plating on one of the validated media listed in the procedure.
• An isolated colony from Brain Heart Infusion (BHI) agar or Nutrient Agar (NA) may also be characterized by this method if the colony was purified from one of the selective agars listed in the cultural method, and/or had previously been confirmed as E. coli.
• Process a positive control consisting of an isolate of the VTEC laboratory strain and a negative control consisting of an isolate of the generic E. coli (non-VTEC) strain along with each set of samples.

Note: To avoid delays, well isolated, typical colonies may be concurrently streaked to BHI, NA or Rainbow agar when picking typical isolates for confirmation as described in MFHPB-10: Isolation of Escherichia coli O157:H7/NM from Foods and Environmental Surface Samples. Suspect E. coli O157 isolates can then be assayed by the VT-PCR from colonies obtained off of the Rainbow, BHI or NA plates.

Procedure

Preparation of cell lysates

Pick a portion of a single suspect isolated colony from Rainbow agar, BHI agar or nutrient agar with a loop and suspend the colony in 200 µL of 1% (v/v) Triton X-100 in a sterile 1.5 mL microfuge tube with a secured cap (e.g. screw-cap); vortex to mix. Set up a lysis buffer negative control tube by dispensing 200 µL of 1% (v/v) Triton X-100 and carry it through the remainder of the procedure. Similarly, set up the positive and negative controls and carry them through the remainder of the procedure.
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Prepare the template for PCR by heating the tubes at 100 °C in a heat block for 10 min. Cool the resulting lysates for 5 min 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.

VT-PCR

Label the reaction tubes.
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Prepare the VT-PCR master mix as described under VT-PCR master mix and dispense 45 µL into 0.2 or 0.5 mL PCR tubes. Add 5 µL of the colony lysate to the corresponding tubes (including the positive and negative control cultures). Also include one control reaction of PCR master mix with 5 µL of water but no template, and one control reaction of PCR master mix with 5 µL of the 1% Triton X-100 devoid of template.
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Insert the tubes in the thermal cycler and proceed with the PCR amplification using the cycling program given under VT-PCR cycling program. If necessary, the PCR products can be stored at 4 °C for up to 24 h or at −20 °C for up to one week.
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After the PCR is completed, analyse the PCR products by FlashGel electrophoresis (follow the manufacturer’s instructions).
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Load 5 µL of the FlashGel 50 bp-1.5 kb DNA marker into the first well of a FlashGel 2.2% DNA cassette. To prepare the PCR product for FlashGel electrophoresis, mix 2 µL of the PCR product with 2 µL of ultrapure or molecular grade water and 1 µL of 5X Flash Gel loading dye. Load the prepared samples into the remaining wells of the FlashGel DNA cassette.
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Run the FlashGel for approximately 6.5 min 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. The IAC may appear faint for VT positive samples.

Interpretation of results

• A sample is considered positive if it is positive for VT1 and/or VT2.
• Samples testing negative for both VT1 and VT2 genes and positive for IAC are considered negative.
• Samples testing negative for both VT1 and VT2 genes and negative for IAC is considered as evidence of PCR inhibition by sample matrix contaminants, failure of one of the detector reagents, or omission of a critical component in the procedure. The results of the run are invalidated and the VT-PCR must be repeated.
• If VT bands are observed in the negative and/or non-template or sterility controls, the results of the run are invalidated and the run must be repeated with precautions taken to eliminate possible sources of contamination.

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 ± 1.0 °C. Similarly, lower temperatures of 30 or 25 may be ± 1.0 °C. However, where higher temperatures are recommended, such as 43 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, unless otherwise specified in the method.

References

• Health Canada, Health Products and Food Branch. Determination of Verotoxin genes in Verotoxigenic Escherichia coli isolates. Compendium of Analytical Methods, Volume 3, Laboratory Procedure MFLP-62, November 2015.
• 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.
• Gauthier, M. and Blais, B.W. 2005. Cloth-based hybridization array system for detection of toxin genes associated with major foodborne pathogenic bacteria. Journal of Rapid Methods & Automation in Microbiology. 13: 243–256.
• 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, rfb O111, and rfbO157. Journal of Clinical Microbiology, 36:598–602.