SOP: Concentration and Detection of Hepatitis A Virus from Contaminated Strawberries by the Pathatrix System and Reverse-Transcriptase Polymerase Chain Reaction

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

Strawberries suspected of contamination with hepatitis A virus (HAV) are washed in a glycine buffer, and the wash is circulated forcefully through a Pathatrix workstation containing non-specific positively charged magnetic beads. The cationic beads bind the negatively charged viral capsids, and the bead–virus complexes are drawn onto a magnet, washed with phosphate-buffered saline (PBS) and resuspended in PBS. Viral RNA is extracted with the QIAamp Viral RNA Mini Kit, amplified by reverse-transcriptase polymerase chain reaction (RT-PCR) with HAV-specific primers, and the amplicons are read on an agarose gel. The method does not differentiate between infectious and non-infectious viruses. In test sensitivity studies, the method consistently detected 1000 PFU of HAV in artificially inoculated strawberries.

2. Apparatus required

• Thermal cycler (Eppendorf Mastercyler Gradient or equivalent)
• Microfuge 22R refrigerated tabletop centrifuge (Beckman Coulter or equivalent)
• Microwave oven or hot plate
• Submarine gel casting tray with buffer reservoir, power pack and comb
• Shortwave UV light table (transilluminator) for visualizing stained DNA in agarose gels
• Photo documentation system (optional, for photographic records), including Polaroid camera (hand-held or fixed), hood and Polaroid 667 film or equivalent
• Adjustable micropipettors (0.5–10, 10–100 and 100–1000 µL) with filtered pipette tips
• Waterbath capable of maintaining 40–50°C (where 37°C is recommended, ±1°C; all other temperatures, ±2°C)
• Freezers capable of maintaining −20°C and −80°C
• Pathatrix or Pathatrix-ULTRA workstation (Matrix Microscience Inc.)
• Magnetic wash tube rack (Matrix Microscience Inc.)
• Timer
• Container for ice
• Vortex mixer
• Pre-sterilized plastic and rubber disposable kits (Matrix Microscience Inc.)
• Microfuge tubes (2.0 and 1.5 mL)
• Thin-wall PCR tubes (0.2 mL; type depends on the thermal cycler model)
• Medium disposable weigh boats
• Stomacher bags with filter

3. Reagents and kits

• Custom magnetic silica particles, cationically/positively charged (Matrix Microscience Inc., product number PCAT50 — no substitutions; the source prints “cationally”)
• Feline calicivirus strain F9 (American Type Culture Collection, product number VR-782 — no substitute)
• QIAamp Viral RNA extraction kit (product number 52904 or 52906 — no substitutions)
• Qiagen OneStep RT-PCR Kit (product numbers 210210 or 210212 — no substitutions)
• Agarose (molecular biology grade)
• DNA ladder, 100 bp (or equivalent)
• Ethidium bromide (molecular biology grade) or SYBR Safe dye (Invitrogen product number S33102 or equivalent)
• RNase inhibitor
• Boric acid (molecular biology grade)
• Tris[hydroxymethyl]aminomethane (e.g. Tris base, molecular biology grade)
• Ethylene diamine tetra acetic acid (EDTA) disodium salt (molecular biology grade)
• Glycine, non-animal source (molecular biology grade)
• Sodium chloride (molecular biology grade)
• NaOH (ACS grade or better)
• HCl (ACS grade or better)
• DNase/RNase-free water (molecular biology grade)
• Phosphate-buffered saline, pH 7.2 (Invitrogen product number 20012-027 or equivalent)
• Glycine buffer — see section 8.1
• 5× Tris-Borate-EDTA (TBE) buffer — see section 8.3
• Ethanol, 95–99%
• Houde/Guévremont primers for HAV (SH-Prot-A and SH-Prot-1) — see section 6.1
• FCV primers for feline calicivirus (CBK-1 and CBK-2) — see section 6.2

Reagent preparations

• Phosphate Buffered Saline (PBS)
• Stock Solution 1
• Sodium phosphate dibasic anhydrous reagent grade (Na₂HPO₄) — 28.4 g
• Sodium chloride reagent grade (NaCl) — 85.0 g
• Distilled water — 1.0 L
• pH approximately 8.2
• Stock Solution 2
• Sodium phosphate monobasic monohydrate reagent grade (NaH₂PO₄·H₂O) — 27.6 g
• Sodium chloride reagent grade (NaCl) — 85.0 g
• Distilled water — 1.0 L
• pH approximately 5.6
• Complete Medium: make 1 to 9 dilutions of aliquots of each stock solution to obtain a final volume of 500 mL of diluted solution 1, and 100 mL of diluted solution 2; by means of a pH meter, titrate the diluted solution 1 to a pH of 7.3–7.4 by adding approximately 65.0 mL of diluted solution 2; the resulting solution is 0.02 M phosphate saline buffer
• Note: do not titrate 0.2 M phosphate buffer to pH 7.3–7.4 and then dilute to 0.02 M strength — this results in a drop in pH of approximately 0.25. Addition of 0.85% salt after pH adjustment also results in a drop of approximately 0.2

4. Procedure

Sample handling

Keep the sample units refrigerated (1–5°C) during storage and transport; analyse them as soon as possible after receipt

The number of samples is determined on the basis of client needs (e.g. data collection or surveys) or investigational purposes (e.g. outbreaks)

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Preparation for analysis

Prepare all buffers and solutions as described in section 8

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Prepare and verify the suitability of all controls — spike each test sample with 10⁵ plaque-forming units (PFU) of the feline calicivirus (FCV) internal processing control; run one negative processing control (uninoculated strawberries purchased locally) in parallel with each set of up to four test samples; HAV and FCV RNA must be available for use as PCR positive controls

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Weigh 25 g of strawberries onto a disposable weigh boat (up to a maximum of four test samples and one negative process control)

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Add 10⁵ PFU of FCV to each test sample (up to a maximum of four test samples) — it is recommended to dilute the viral stock in PBS so the inoculation can be done in a 100 µL volume

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Concentration of virus with the Pathatrix workstation

The manufacturer provides a video demonstration of the Pathatrix-ULTRA workstation — the method recommends viewing it before processing (manufacturer name: Matrix Microscience Inc.)

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Switch on the Pathatrix workstation

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Transfer each 25 g sample of strawberries into a separate stomacher bag

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Add 225 mL of glycine buffer to each stomacher bag and shake the samples by hand for about 30 s

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Add 1.55 mL of 1 N HCl to the sample plus buffer in the stomacher bag and shake by hand for about 30 s

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Place each stomacher bag into a separate incubation pot on the Pathatrix machine

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Aseptically remove the sterile disposable kit from its bag; orient the pot lid so the tubing extends from the centre and left of the lid (the nine o’clock position)

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Hold the wash vessel in the left hand and place it into the holder; release the phase and, using both hands, insert the pot lid into the stomacher bag; secure the phase within the phase holder and locate the elution pipette under the retainer

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Load the inlet tubing into the pump head, taking care not to trap the tubing when closing the pump head

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Vortex the vial of capture beads for a minimum of 10 s immediately before dispensing — make sure the beads are fully resuspended

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Disconnect the large tubing from the front of the valve that is also connected to the wash vessel; aseptically add 50 µL of capture beads into the tubing; reconnect the large tubing to the front valve

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When loading more than one sample, vortex the capture beads each time before dispensing

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Fill each wash vessel with 100 mL of PBS

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Set the temperature of each unit to 20–25°C and set the timer to 30 min

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Press “RUN” — recirculation starts immediately; push the pot lid down firmly (the timer only begins when the test temperature is reached)

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When the run is complete, the system displays the wash-ready symbol

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Twist the tab on the side of the plunger on the front valve until it breaks off; using the first finger and thumb, press the plunger down until it reaches a natural stop (excessive force is not required)

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Press “RUN” on the key pad to start the wash step; press “RUN” a second time to stop the pump before air enters the phase

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Twist the tab on the side of the plunger on the rear valve until it breaks off; using the thumb and first finger, press the plunger until it reaches a natural stop (excessive force is not required)

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Open the pump head and carefully remove the phase from the magnet

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Using the pipette attached to the rear valve, gently flush the wash buffer over the phase a minimum of 5 times

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Remove the wash vessel lid, with its internal tube still attached, from the wash vessel and let the liquid drain back into the wash vessel

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Discard the stomacher bag and disposable kit as biohazardous waste; place the flat lid provided on the wash vessel

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Put the wash vessel into the wash vessel rack and leave it standing at room temperature for 5 min so the beads are drawn to the magnet

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Aseptically remove all of the liquid from the wash vessel, taking care not to remove any of the beads

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Resuspend the beads by a brief vortex in 140 µL of PBS and transfer each sample to a sterile 1.5 mL microfuge tube

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RNA extraction with the QIAamp Viral RNA Mini kit

All reagents in this section are provided in the kit, except cold ethanol. The extraction follows the manufacturer’s kit-manual protocol for nucleic acids from viruses. Once carrier RNA has been added to the AVL buffer, store it at 4°C. The AVL buffer must be completely thawed so any precipitate dissolves before use

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The samples (beads in 140 µL of PBS) are ready in 1.5 mL microfuge tubes; if HAV and FCV RNA positive controls are needed, 140 µL of viral stock may be placed in a 1.5 mL microfuge tube and processed in parallel with the test samples

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Add 560 µL of buffer AVL to each sample

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Vortex the tubes for 2 s

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Incubate the samples for 10 min at room temperature (15–25°C)

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Add 560 µL of ethanol to the samples

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Vortex the tubes

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Transfer 630 µL of the solution to a QIAamp spin column from the kit

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Close the caps and centrifuge at 6,000 × g (8000 rpm) for 1 min (each centrifugation in this section is carried out at room temperature)

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Transfer each spin column to a clean 2 mL collection tube from the kit; discard the used collection tubes

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Transfer the remaining 630 µL of each solution to its spin column; centrifuge at 6,000 × g (8000 rpm) for 1 min

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Transfer each spin column to a clean 2 mL collection tube; discard the used collection tubes

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Add 500 µL of Buffer AW1 to each spin column and close the lids; centrifuge at 6,000 × g (8000 rpm) for 1 min

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Transfer each spin column to a clean 2 mL collection tube; discard the used collection tubes

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Add 500 µL of Buffer AW2 to each spin column and close the lids; centrifuge at 20,000 × g (14,000 rpm) for 3 min

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Place each spin column in a 1.5 mL microfuge tube; discard the used collection tubes

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Add 60 µL of Buffer AVE to each spin column; close the caps and incubate at room temperature for 1 min

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Centrifuge at 6,000 × g (8000 rpm) for 1 min

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Discard the spin columns and close the lids of the 1.5 mL microfuge tubes

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Use the RNA directly in RT-PCR, or store at −80°C until needed

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RT-PCR for hepatitis A virus

Screen each sample for HAV RNA and FCV RNA in two separate reactions (no multiplexing) — one set of hepatitis A primers (section 6.1) and one set of feline calicivirus primers (section 6.2) — using the Qiagen OneStep RT-PCR kit

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Add 5.0 µL of the sample RNA to 20.0 µL of RT-PCR reaction mixture (section 8.2)

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Set up a negative control by adding 5.0 µL of RNase-free water to 20.0 µL of the same RT-PCR reaction mixture; for positive controls, add 5.0 µL of HAV or FCV RNA (as appropriate) previously confirmed as positive in other experiments to 20.0 µL of the same mixture

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Place the microfuge tubes in the thermal cycler and amplify according to the program in section 7

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After RT-PCR is complete, analyse the PCR product by agarose gel electrophoresis (next step); if necessary, the amplicons can be stored at 4°C until analysis

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Agarose gel electrophoresis

Prepare a 2.0% (w/v) agarose gel in 0.5× TBE buffer — dissolve by stirring on a hot plate or microwaving 1–2 min on high power, until completely clear with no particles in suspension

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Cool the agarose to about 40–50°C and add 2.5 µL of ethidium bromide solution (10 mg/mL) or SYBR Safe dye; mix gently without forming bubbles

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Note — adding ethidium bromide or SYBR Safe to the gel is optional if the gel is submerged in an EtBr or SYBR Safe solution after migration; ethidium bromide IS A POTENT MUTAGEN — wear nitrile gloves when handling it

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Pour into a gel tray sealed with tape (or as provided by the manufacturer), avoiding bubble formation or trapping; add a well-forming comb and let the gel set for 20–30 min

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Prepare the samples: in clean microfuge tubes, mix 2 µL of tracking dye (5× loading buffer) with 10 µL of PCR product

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When the gel has set, remove the comb, place the tray in the electrophoresis apparatus and fill the reservoir with 0.5× TBE buffer so the gel is covered to a depth of 4 mm; gently pipette about 12 µL of each sample into the wells of the submerged gel; pipette a DNA molecular-size marker (e.g. 100 bp ladder DNA) into an empty well; include positive, negative and reagent controls

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Connect the power supply with the cathode (−, black) at the top (near the sample wells) and the anode (+, red) at the bottom (far end) of the gel; apply about 135 volts for about 30 min, or until the tracking dye has spread about two-thirds of the gel length (voltage and migration time may be changed according to the power supply used)

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Remove the gel and visualize the DNA bands by exposure to shortwave ultraviolet light on a transilluminator; photograph the gel on Polaroid 667 film for analysis and record-keeping, or use a digital processing system

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Note — if ethidium bromide was not added directly to the gel, remove the gel from the tray and stain the DNA by placing it in ethidium bromide solution (10 µg/mL) for 10 min; lift the gel out with a gel scoop, rinse briefly with tap water, and visualize under UV light; UV LIGHT CAN CAUSE EYE DAMAGE — wear safety goggles

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Reading the PCR results

The amplicon generated by the HAV primers and the FCV primers are double-stranded DNA fragments of 172 bp and 218 bp, respectively — a positive PCR test shows a band specific to the targeted gene sequence, appearing as an intense band on the EtBr- or SYBR Safe-stained gel; verify the band size against the 100 bp ladder run on the same gel

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A negative PCR test normally shows no visible bands; any sample giving bands that do not match the expected amplicon (non-specific amplification) is considered negative — such bands are extremely rare

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The targeted positive control must show its specific band — without it, the test is invalid and the samples are re-analysed

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A band matching the positive control appearing in the negative or reagent control indicates PCR mixture contamination — the whole batch is suspect and is discarded; the samples are re-analysed with a new reagent batch

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Any test sample showing a distinct band matching its positive control is considered PRESUMPTIVE POSITIVE — the PCR product is confirmed by sequence analysis

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IMPORTANT — amplicons from presumptive positives are sent to a reference laboratory for sequencing, along with the food extracts (if available), to confirm the analysis

5. Interpretation of results

• A positive test shows a 172 bp band (HAV primers) or a 218 bp band (FCV primers) matching the positive control against the 100 bp ladder.
• Non-specific bands do not count as presumptive positive.
• A missing positive-control band invalidates the test — re-analyse.
• A band in the negative or reagent control means contamination — discard the batch and re-analyse with new reagents.
• A distinct band matching the positive control is a PRESUMPTIVE POSITIVE — confirm by sequencing at a reference laboratory.

6. Primer sequences

• HAV (172 bp fragment, based on the protease coding region 2A of HAV):
• SH-Prot-A (forward, 20 bases, positions 3378–3397): 5′-ATG GAT GCT GGR GTT CTT AC-3′
• SH-Prot-1 (reverse, 22 bases, positions 3529–3550): 5′-ART TGG CAG CAA TTT CTT CAA G-3′
• Positions refer to the reference HAV strain HM-175 wild type (GenBank accession NC_001489).
• FCV (218 bp fragment; primers designed in-house by Dr. César Bin Kingombe, Health Canada):
• CBK-1 (upstream, 20 bases): 5′-GGA GGC GCG ATC TTC AGT AT-3′
• CBK-2 (downstream, 20 bases): 5′-GCA TAA CTC GTC GGA GGT GT-3′
• Degenerate-base codes: R = A/G; Y = C/T; M = A/C; K = G/T; S = C/G; W = A/T; H = A/C/T; B = C/G/T; V = A/C/G; D = A/G/T; N = A/C/G/T; X = A/C/G/T.
• Oligonucleotide primer synthesis can usually be contracted out to a local university, or many biotechnology firms offer a custom synthesis service.

7. Temperature cycling program (HAV and FCV primers)

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

• Step 1 — Reverse transcription: 30 min at 50°C
• Step 2 — Initial PCR activation: 15 min at 95°C
• Step 3 — 40 cycles of: 3.1 Melting, 45 s at 94°C; 3.2 Annealing, 45 s at 53°C; 3.3 Extension, 45 s at 72°C
• Step 4 — Final elongation: 10 min at 72°C

The initial activation step inactivates the HotStarTaq DNA polymerase inhibitors and the Omniscript and Sensiscript reverse transcriptases and denatures the cDNA template. Thermal cyclers other than the models stated may alter PCR performance — the user may need to optimize the cycling parameters for a different model.

8. Reagent preparations

• 8.1 Glycine buffer, pH 9.0 (0.05 M glycine, 0.14 M NaCl): 15.01 g glycine; 32.73 g NaCl; 8 mL Tween 20 (all molecular biology grade). Add to 3.5 L of distilled water, dissolve with a magnetic stirrer, adjust the pH to 9.0, complete to 4.0 L and filter-sterilize. Stable for 9 months at 4°C.
• 8.2 Qiagen OneStep RT-PCR mix (batch for 20 reactions; stock solutions stored at −20°C): 200.0 µL RNase-free water; 100.0 µL of 5× OneStep RT-PCR Buffer (final 1×); 20.0 µL dNTP mix (10 mM each dNTP; final 400 µM); 30.0 µL of each primer (10 µM per primer; final 0.6 µM) — SH-Prot-A forward with SH-Prot-1 reverse, or CBK-1 forward with CBK-2 reverse (one primer set per tube); 20.0 µL of enzyme mix. Dispense 20.0 µL of mix per tube; add 5.0 µL of template RNA. All reagents, DNase/RNase-free water, pipette tips and other materials contacting samples or RT-PCR reagents must be sterile or autoclaved; prepare all reagents in a laminar-flow cabinet that has never been exposed to HAV or HAV PCR products; prepare the mix on ice or on a refrigerated rack to avoid non-specific amplification.
• 8.3 5× Tris-Borate-EDTA (TBE) buffer (or commercially available): 54.0 g Tris base; 27.5 g boric acid; 3.75 g EDTA disodium (all molecular biology grade). Add distilled water to 800 mL, dissolve, complete to 1.0 L. Use at a 1:10 dilution (0.5× TBE). The 0.5× buffer pH should be 8.3 — do not adjust.
• 8.4 Tracking dye / loading buffer (10× Orange G, or commercially available): 0.025 g Orange G (sodium salt), 0.25% (w/v); 4 mL glycerol, 40% (w/v); 6 mL sterile distilled water (DNase/RNase-free). Mix thoroughly; sterilize by autoclaving at 121°C for 15 min or filter-sterilize; store in 1.0 mL aliquots at −20°C. Stable for 12 months at −20°C (the source prints “binding buffer”). Store the working solution at 4°C.
• 8.5 DNA molecular-size marker (commercially available): the 100 bp ladder DNA provides a useful range of DNA fragment sizes and facilitates sizing of the PCR amplicons.

9. Limitations

• This method will not differentiate between infectious and non-infectious viruses.

10. Precautions

• The laboratory supervisor must ensure the analysis is done in accordance with ISO/IEC 17025:1999 (or latest version) — “General requirements for the competence of testing and calibration laboratories”.
• Where 37°C is recommended, the waterbath may be at 37°C ±1°C; for all other temperatures it may be ±2°C.
• Ethidium bromide is a potent mutagen — handle with nitrile gloves.
• UV light can cause eye damage — wear safety goggles.

References

• Health Canada, Compendium of Analytical Methods, Volume 5 — Laboratory Procedure OPFLP-09, March 2010: “Concentration and detection of hepatitis A virus from contaminated strawberries by the Pathatrix system and reverse transcription polymerase chain reaction.”
• QIAamp Viral RNA Mini kit insert, January 1999.
• Qiagen OneStep RT-PCR kit insert, May 2002.
• Matrix Microscience Inc., accessed on July 17th, 2006.
• Papafragkou, E., Plante, M., Mattison, K., Bidawid, S., Karthikeyan, K., Farber, J. M. and Jaykus, L.-A. 2007. Rapid and sensitive detection of hepatitis A virus in representative food matrices. Journal of Virological Methods, 147: 177–187.
• Guévremont, E., Brassard, J., Houde, A., Simard, C. and Trottier, Y.-L. 2006. Development of an extraction and concentration procedure for the detection of hepatitis A virus and norovirus from green onions by RT-PCR. Journal of Virological Methods, 134: 130–135.