SOP: Concentration of Norovirus Genogroup II and Hepatitis A Virus from Contaminated Green Onions and Their Detection Using the Reverse-Transcriptase Polymerase Chain Reaction

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

Virus particles are washed off the surface of fresh green onions by repeated pipetting with a pH 9.5 tryptose phosphate broth–glycine buffer, and the viral load is concentrated by polyethylene glycol (PEG) precipitation. Viral RNA is extracted and purified by a TRIzol–chloroform step followed by a poly(dT) magnetic-bead step; the beads capture the polyadenylated (poly-A) messenger RNA of noroviruses and hepatitis A virus. The final extract is amplified by reverse-transcriptase polymerase chain reaction (RT-PCR), which produces a DNA fragment whose size depends on the primer set used — norovirus genogroup II or hepatitis A virus.

2. Apparatus required

• Thermal cycler (Eppendorf Mastercycler Gradient or equivalent)
• Microwave oven or hot plate
• Submarine gel casting tray and buffer reservoir, power pack and an appropriate comb
• Shortwave UV light table (transilluminator) for viewing 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 covering 0.5 to 10 µL, 10 to 100 µL and 100 to 1000 µL, with specific sterile filtered pipet tips
• Standard heatblocks (VWR Scientific products or equivalent) holding 1.5 mL microfuge tubes and maintaining 65 °C and 90 °C
• Platform balance with 0.01 g resolution
• Vortex mixer
• Microfuge tubes, 2.0 mL and 1.5 mL capacity
• Sorvall RC-5B Plus refrigerated centrifuge with SS-34 rotor (8 × 50 mL polypropylene copolymer Oak Ridge tubes) or equivalent
• 50 mL centrifugation tubes (polypropylene copolymer Oak Ridge or polypropylene)
• IEC DPR-6000 refrigerated centrifuge or equivalent
• IEC DPR-6000 rotors for 15 mL tubes or equivalent
• 15 mL polypropylene centrifugation tubes (Sarstedt product number 62.554.002 or better; the source prints “Sarstead”)
• Dynal magnetic bead attractor, model MPC-S (product number 120.20, no substitution)
• Dynal Biotech Sample Mixer (product number 947.01 or equivalent)
• Sorvall Legend RT refrigerated centrifuge with Heraeus rotor number 3332
• Thin-wall PCR tubes, 0.2 mL or 0.5 mL capacity (depending on thermal cycler model)
• Timer
• Container for ice
• Magnetic stirrer
• Freezers maintaining −20 °C and −70 °C
• 5 mL and 25 mL serological pipettes
• Pipet-Aid or equivalent
• 150 mm Petri dish
• Filtration unit (Millipore Steritop product number SCGPT05RE or equivalent)

3. Reagents and kits

• QIAGEN OneStep RT-PCR Kit (product numbers 210210 or 210212, no substitution)
• Dynabeads-oligo(dT)25 (Cat. No. 610.05, no substitution)
• Binding buffer (prepared per section 8.3)
• Washing buffer (prepared per section 8.4)
• Tryptose phosphate broth–glycine buffer (TPB-glycine buffer; prepared per section 8.1)
• TRIzol Reagent, Invitrogen (Cat. No. 15596-018/-026, no substitution)
• Chloroform (HPLC grade or better)
• Isopropanol (HPLC grade or better)
• Cold 75% ethanol in DNase/RNase-free water (stored at −20 °C)
• Houde/Guévremont primers (SH-Prot-A and SH-Prot-1) for hepatitis A virus (sequences per section 6.1)
• Kageyama primers (COG2F and COG2R) for norovirus genogroup II (sequences per section 6.1)
• Monroe primers (431, 432, 433 and 434) for norovirus genogroups I and II (sequences per section 6.1)
• Agarose (molecular biology grade)
• Orange G (for tracking dye in loading buffer, per section 8.7)
• DNA Ladder 50 bp (or equivalent)
• RNase inhibitor
• Boric acid (molecular biology grade)
• Tris(hydroxymethyl)aminomethane (e.g., Tris base, molecular biology grade)
• Ethylene diamine tetraacetic acid (EDTA), disodium salt (molecular biology grade)
• Water, DNase/RNase-free (molecular biology grade)
• Polyethylene glycol (PEG) 8000 (molecular biology grade)
• Sodium chloride (NaCl) (molecular biology grade)
• Dynabeads Reconditioning Solution: sodium hydroxide (NaOH) (ACS grade or better)
• Dynabeads Storage Buffer: Tris(hydroxymethyl)aminomethane (molecular biology grade), EDTA disodium salt (molecular biology grade), Tween 20 (molecular biology grade), sodium azide (ACS or better)

Reagent preparations

• Cold 75% Ethanol in DNase/RNase-free Water
• To prepare: mix 75 mL of ethanol with 25 mL of DNase/RNase-free water; store at −20 °C

4. Procedure

Sample handling

Determine the number of sample units from the client’s needs (e.g., data collection or surveys) or from the investigation purpose (e.g., an outbreak)
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Keep the sample units refrigerated (1 °C to 5 °C) during storage and transport
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Analyse the sample units as soon as possible after receipt at the laboratory

Preparing for analysis

Prepare all buffers and solutions as described in section 8
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Prepare and verify all controls; run a positive and a negative control in the RT-PCR test, specific to the primer set in use (norovirus genogroup II and hepatitis A virus)
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Bring all extraction reagents to room temperature (23 °C ± 3 °C) before use (i.e., the TRIzol reagent)
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Carry out the virus extraction and concentration steps under a biological containment hood wearing a mask, sleeve protectors and double nitrile gloves — additional precautions apply when handling green onions that may carry noroviruses or other viruses

Virus elution and concentration

Cut a 25 g portion of green onions into 2–3 cm pieces in a sterile Petri dish with a sterile pair of scissors, using mainly the green leaf part
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Keep the green onions refrigerated whenever possible — virus integrity is better conserved at colder temperatures
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Elute viruses from the surface of the cut green onions by pipetting ten times with an initial 20 mL volume of 2.9% (w/v) tryptose phosphate broth with 6% (w/v) glycine at pH 9.5 (23 °C ± 3 °C), to detach any potential viruses
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Transfer the residual washing solution (about 16–17 mL) from the cut green onions into an Oak Ridge tube
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Add an equal volume of 16% PEG with 0.525 M NaCl, to reach a final concentration of 8% (v/v)
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Invert the tubes 7 times and incubate overnight in a bucket of ice (18 hours ± 1 hour) at 4 °C
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Centrifuge the Oak Ridge tube at 12,000 × g for 30 minutes at 4 °C
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Discard the supernatant, leaving the last 2–3 mL so the pellet is not lost
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Add 2 mL of TRIzol reagent to the pellet — TRIzol contains phenol; handle carefully and dispose of as hazardous waste
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Vortex the Oak Ridge tube for 30 seconds and incubate 5 minutes at room temperature (23 °C ± 3 °C)
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Add 0.480 mL of chloroform to the Oak Ridge tube, vortex for 30 seconds, and incubate 5 minutes at room temperature (23 °C ± 3 °C)
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Transfer the whole suspension into three 2 mL micro-tubes and centrifuge at 12,000 × g for 10 minutes at 4 °C
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Recover the upper aqueous phase, pool it into a 15 mL centrifugation tube, add 0.5 mL of isopropanol, invert by hand 5 times, and incubate 5 minutes at room temperature
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Centrifuge the 15 mL tube at 5,000 × g for 5 minutes at 4 °C, then remove and discard the supernatant
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Wash the surface of the RNA pellet with 1 mL of cold 75% ethanol in DNase/RNase-free water (stored at −20 °C) without breaking the pellet
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Remove the 1 mL of cold ethanol gently after each wash and let the pellet dry 10 to 30 minutes before adding water to it
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Add 400 µL of DNase/RNase-free water to dissolve the RNA pellet completely and transfer the RNA into a 1.5 mL microtube — the total RNA concentrate can be stored at −70 °C or used immediately
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Add 400 µL of binding buffer, then vortex for 30 seconds
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Incubate at 65 °C for 2 minutes in a heatblock, then cool on ice
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Mix the bottle of Dynabeads-oligo(dT)25 thoroughly by inverting by hand for 30 seconds
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Transfer 100 µL of Dynabeads-oligo(dT)25 into a 2.0 mL microtube, place the tube in the magnetic bead attractor for 60 seconds with the magnet slide in, then discard the solution by gentle pipetting without disrupting the beads; remove the magnet slide — insert the slide with its protruding edge downwards and the tape side facing the tubes
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Add 100 µL of binding buffer to the 2.0 mL microtube and mix gently; insert the magnet slide into the attractor for 60 seconds, discard the binding buffer with a pipette, and remove the slide
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Add 400 µL of binding buffer and the 800 µL of total RNA to the 2.0 mL microtube containing the Dynabeads-oligo(dT)25
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Invert the 2.0 mL microtube twice by hand and rotate on the sample mixer at 8 rpm for 5 minutes — if solution lodges under the cap between washing steps, microcentrifuge for 2 seconds before using the magnetic bead attractor
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Insert the magnet slide into the attractor for 60 seconds, remove the binding buffer from the 2.0 mL microtube, and remove the slide — if bead separation is incomplete, prolong the incubation until the beads separate properly
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Wash the magnetic beads by adding 500 µL of washing buffer, mixing by 5 inversions; insert the magnet slide into the attractor for 60 seconds, then remove and discard the washing buffer; remove the slide
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Repeat the washing step two more times
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Remove the last traces of the final wash after microcentrifuging for 2 seconds, with the magnet slide in the attractor
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Resuspend the magnetic beads in 25 µL of RNase/DNase-free water and elute the poly-A viral RNA from the beads at 90 °C for 2 minutes in a heatblock
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Separate the heated solution from the beads in the magnetic bead attractor
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Transfer the solution (poly-A RNA) to a 0.5 mL or 1.5 mL tube
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Add 20 units of RNase inhibitor to the solution
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Store the tube at −70 °C

Screening RT-PCR for norovirus genogroup II or hepatitis A virus

Screen the poly-A RNA of each sample with the norovirus primers and hepatitis A virus primers described in section 6.1, using the QIAGEN OneStep RT-PCR kit — validation data showed the COG (Kageyama) primers give the best analytical sensitivity, while the Monroe primers are less sensitive but amplify a broader range (including caliciviruses outside genogroups I and II) and are widely used for molecular characterization of norovirus strains by sequencing
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Add 4.0 µL of the poly-A RNA to 16.0 µL of RT-PCR reaction mixture (per section 8.5)
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Place the microfuge tubes in a thermal cycler and amplify according to the temperature cycling program for the primer set in use (section 7)
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After RT-PCR, analyse the PCR product by agarose gel electrophoresis below; amplicons may be kept at 4 °C for up to one week, or at −20 °C for longer, until analysis

Agarose gel electrophoresis

Prepare a 1.5% (w/v) agarose gel in 0.5× TBE (Tris-Borate-EDTA) buffer; dissolve the agarose on a hot plate or in a microwave for 1 to 2 minutes at high power until completely dissolved (clear liquid, no particles)
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Cool the agarose to about 45 °C and add ethidium bromide (EtBr) concentrate to a final concentration of 0.5 µg/mL in the gel; mix gently without forming bubbles — adding EtBr to the gel is optional if the gel is stained in EtBr solution after migration; EtBr is a potent mutagen, wear nitrile gloves
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Pour into a gel tray, avoiding bubbles, add a well-forming comb, and let the gel solidify for 20 to 30 minutes
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In clean microfuge tubes, mix 2 µL of Orange G tracking dye (10× loading buffer) with 20 µL of PCR product
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Once set, remove the comb, place the tray in the electrophoresis apparatus, and cover the gel with 0.5× TBE buffer
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Pipette about 18 µL of each sample into the wells of the submerged gel; pipette a DNA molecular size marker (e.g., 50 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; apply about 100 volts for about 30 minutes, or until the Orange G tracking dye has travelled about three-quarters of the gel length — voltage and migration time can be changed for the power supply in use
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Remove the gel from the tray and view the DNA bands under shortwave ultraviolet light on a transilluminator; gels may be photographed on Polaroid 667 film or with a digital processing system — UV light can cause eye damage, wear safety goggles
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Where EtBr was not added to the gel, remove the gel from the tray and stain the DNA in ethidium bromide solution (0.5 µg/mL) for 30 to 45 minutes; lift the gel out with a gel scoop, rinse briefly with tap water, and view under UV light

Regenerating the Dynabeads-oligo(dT)25

The Dynabeads-oligo(dT)25 may be reused up to four times without loss of yield
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Resuspend the used Dynabeads-oligo(dT)25 (original volume 200 µL) in 200 µL of Reconditioning Solution and transfer the suspension to a new RNase-free tube
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Incubate at 65 °C in a heatblock for 2 minutes
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Place the tube in the Dynal MPC-S attractor for at least 30 seconds and remove the supernatant
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Wash twice in Reconditioning Solution by repeating the incubation and separation steps twice
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Resuspend the Dynabeads-oligo(dT)25 in 200 µL of storage buffer Oligo(dT)25
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Place the tube on the magnet for at least 30 seconds and remove the supernatant
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Repeat the resuspend-and-separate steps three times
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Resuspend the Dynabeads-oligo(dT)25 with storage buffer Oligo(dT)25 to visually match the concentration of a new, unregenerated lot — the beads are now reconditioned and ready for another mRNA isolation; store in storage buffer Oligo(dT)25 at 2–8 °C
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Do not mix regenerated Dynabeads-oligo(dT)25 with the original stock suspension

5. Interpretation of results

• The amplicons generated by the COG2F/R, Monroe and SH-Prot-1/SH-Prot-A primers are double-stranded DNA fragments of 98 bp, 213 bp and 172 bp respectively; a positive PCR test yields the DNA fragment specific to the targeted gene sequence, seen as a band on an EtBr-stained agarose gel, and its size is checked against the DNA molecular size marker run on the same gel
• A negative PCR test normally shows no visible bands on an EtBr-stained gel; a sample giving bands that do not match the expected amplicon size (non-specific amplification) is also considered negative
• A specific band must appear for the targeted norovirus positive control and the hepatitis A virus control — absence of a positive-control band invalidates the test, and the samples are re-analysed
• A band matching the positive control appearing in the negative or reagent control with the norovirus or hepatitis A virus primers indicates contamination of the PCR reaction mixture; the whole batch is suspect and is discarded, and the samples are re-analysed with fresh reagents
• A test sample showing a distinct band with the norovirus or hepatitis A virus primers, matching its positive control, is considered presumptive positive; purify (or purify and clone) the PCR product before sequencing to confirm the strain of norovirus or hepatitis A found in the sample
• IMPORTANT: send amplicons from presumptive positives to a reference laboratory for sequencing, together with the food extracts where available, for confirmation of the analysis

6. Primer sequences

Screening PCR uses oligonucleotide primers based on the most conserved region of the norovirus genome.

• Kageyama primers for norovirus genogroup II (98 bp fragment):
• COG2F — forward — 26 bases — 5′-CAR GAR BCN ATG TTY AGR TGG ATG AG-3′
• COG2R — reverse — 21 bases — 5′-TCG ACG CCA TCT TCA TTC ACA-3′
• Monroe primers for norovirus genogroups I and II (213 bp fragment):
• 431 — forward — 20 bases — 5′-TGG ACI AGR GGI CCY AAY CA-3′
• 432 — forward — 20 bases — 5′-TGG ACI CGY GGI CCY AAY CA-3′
• 433 — reverse — 21 bases — 5′-GAA YCT CAT CCA YCT GAA CAT-3′
• 434 — reverse — 21 bases — 5′-GAA SCG CAT CCA RCG GAA CAT-3′
• Houde/Guévremont primers for hepatitis A virus (172 bp fragment):
• SH-Prot-A — forward — 20 bases — 5′-ATG GAT GCT GGR GTT CTT AC-3′
• SH-Prot-1 — reverse — 22 bases — 5′-ART TGG CAG CAA TTT CTT CAA G-3′

Standard mixed-base definitions:

• 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 be contracted to a local university or a biotechnology custom-synthesis service; for better results use primers purified by high-pressure/performance liquid chromatography (HPLC).

7. Temperature cycling programs

For all norovirus primers specific for genogroup II (Monroe and Kageyama primers):

• Reverse transcription — 30 minutes at 50 °C
• Initial PCR activation — 15 minutes at 95 °C (activates the HotStarTaq DNA polymerase; the Omniscript and Sensiscript reverse transcriptases are inactivated and the cDNA template is denatured)
• 40 cycles of:
• Melting — 45 seconds at 94 °C
• Annealing — 30 seconds at 52 °C
• Extension — 45 seconds at 72 °C
• Final elongation — 10 minutes at 72 °C

For primers specific for hepatitis A virus (the source prints “Hepatits A Virus”):

• Reverse transcription — 30 minutes at 50 °C
• Initial PCR activation — 15 minutes at 95 °C (activates the HotStarTaq DNA polymerase; the Omniscript and Sensiscript reverse transcriptases are inactivated and the cDNA template is denatured)
• 40 cycles of:
• Melting — 45 seconds at 94 °C
• Annealing — 45 seconds at 53 °C
• Extension — 45 seconds at 72 °C
• Final elongation — 10 minutes at 72 °C

Thermal cyclers other than the models stated may alter PCR performance; optimize the cycling parameters for the model in use.

8. Reagent preparations

Tryptose Phosphate Broth–Glycine Buffer (TPB-glycine buffer), pH 9.5

• 29 g tryptose phosphate broth (molecular biology grade)
• 60 g glycine (molecular biology grade)
• Add distilled water to 800 mL, dissolve, adjust the pH to 9.5, and complete to 1.0 L
• Filter-sterilize the solution through a 0.22 µm filter unit
• The final pH must be 9.5 ± 0.1 after filter sterilization; the buffer is stable for 9 months at 4 °C

16% Polyethylene Glycol (PEG) with 0.525 M NaCl

• 80 g PEG 8000 (molecular biology grade)
• 15.34 g NaCl (molecular biology grade)
• Add distilled water to 500 mL, dissolve, and autoclave at 121 °C for 15 minutes
• Stable for 9 months at 4 °C

Binding Buffer

• 20 mM Tris-HCl, pH 7.5 (molecular biology grade, commercially available)
• 1.0 M lithium chloride (LiCl) (molecular biology grade, commercially available)
• 2 mM EDTA (molecular biology grade, commercially available)
• To prepare 50 mL from 0.1 M Tris-HCl, 8 M LiCl and 0.5 M EDTA stock solutions:
• 10 mL of 0.1 M Tris-HCl, pH 7.5
• 6.25 mL of 8.0 M LiCl
• 0.200 mL of 0.5 M EDTA
• 33.55 mL of sterile distilled water (DNase/RNase-free) to 50 mL
• Mix all reagents in a sterile 50 mL centrifugation tube and aliquot into smaller volumes for storage; stable for 12 months at room temperature (23 °C ± 3 °C)

Washing Buffer

• 10 mM Tris-HCl, pH 7.5 (molecular biology grade, commercially available)
• 0.15 M lithium chloride (LiCl) (molecular biology grade, commercially available)
• 1 mM EDTA (molecular biology grade, commercially available)
• To prepare 50 mL from 0.1 M Tris-HCl, 8 M LiCl and 0.5 M EDTA stock solutions:
• 5 mL of 0.1 M Tris-HCl, pH 7.5
• 0.938 mL of LiCl (the source prints “0.938 mL of 1.0 M LiCL” though the calculation above uses 8 M LiCl)
• 0.100 mL of 0.5 M EDTA
• 43.962 mL of sterile distilled water (DNase/RNase-free) to 50 mL
• Mix all reagents in a sterile 50 mL centrifugation tube and aliquot into smaller volumes for storing; stable for 12 months at room temperature (23 °C ± 3 °C)

QIAGEN OneStep RT-PCR reaction mixtures (20-reaction batch recipes)

Keep all reagents, DNase/RNase-free water, pipet tips and other materials that touch samples or RT-PCR reagents sterile or autoclaved to remove DNases and other contaminants. Prepare all reagents in a laminar flow cabinet never exposed to noroviruses, norovirus PCR products, hepatitis A virus or HAV PCR products. To avoid non-specific amplification, keep all reagents on ice or on a cooled rack while preparing the mix. Stock solutions are stored at −20 °C.

Monroe primers (volumes for 20 tubes; per tube in parentheses; final concentration):

• RNase-free water — 112.0 µL (5.6 µL)
• 5× QIAGEN OneStep RT-PCR Buffer — 80.0 µL (4 µL; 1×)
• dNTP Mix, 10 mM of each dNTP — 16.0 µL (0.8 µL; 400 µM)
• Primer 431 (forward), 10 µM per primer — 24.0 µL (1.2 µL; 0.6 µM)
• Primer 432 (forward), 10 µM per primer — 24.0 µL (1.2 µL; 0.6 µM)
• Primer 433 (reverse), 10 µM per primer — 24.0 µL (1.2 µL; 0.6 µM)
• Primer 434 (reverse), 10 µM per primer — 24.0 µL (1.2 µL; 0.6 µM)
• QIAGEN OneStep RT-PCR Enzyme Mix — 16.0 µL (0.8 µL)
• Total — 320 µL; distribute 16 µL per tube; template 4 µL per tube

Kageyama primers for genogroup II (volumes for 20 tubes; per tube in parentheses; final concentration):

• RNase-free water — 160.0 µL (8 µL)
• 5× QIAGEN OneStep RT-PCR Buffer — 80.0 µL (4 µL; 1×)
• dNTP Mix, 10 mM of each dNTP — 16.0 µL (0.8 µL; 400 µM)
• Primer COG2F (forward), 10 µM per primer — 24.0 µL (1.2 µL; 0.6 µM)
• Primer COG2R (reverse), 10 µM per primer — 24.0 µL (1.2 µL; 0.6 µM)
• QIAGEN OneStep RT-PCR Enzyme Mix — 16.0 µL (0.8 µL)
• Total — 320 µL; distribute 16 µL per tube; template 4 µL per tube

Houde/Guévremont primers for HAV (volumes for 20 tubes; per tube in parentheses; final concentration):

• RNase-free water — 160.0 µL (8 µL)
• 5× QIAGEN OneStep RT-PCR Buffer — 80.0 µL (4 µL; 1×)
• dNTP Mix, 10 mM of each dNTP — 16.0 µL (0.8 µL; 400 µM)
• Primer SH-Prot-A (forward), 10 µM per primer — 24.0 µL (1.2 µL; 0.6 µM)
• Primer SH-Prot-1 (reverse), 10 µM per primer — 24.0 µL (1.2 µL; 0.6 µM)
• QIAGEN OneStep RT-PCR Enzyme Mix — 16.0 µL (0.8 µL)
• Total — 320 µL; distribute 16 µL per tube; template 4 µL per tube

5× Tris-Borate-EDTA (TBE) buffer

• 54.0 g Tris base (molecular biology grade)
• 27.5 g boric acid (molecular biology grade)
• 3.75 g EDTA, disodium (molecular biology grade)
• Add distilled water to 800 mL, dissolve, and complete to 1.0 L
• Use at a 1:10 dilution (0.5× TBE) in distilled water; the 0.5× buffer should be at pH 8.3 — do not adjust the pH

Tracking dye / loading buffer (10× Orange G)

• 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 minutes or by filter sterilization through a 0.22 µm filter unit; store in 1.0 mL aliquots at −20 °C; stable for 12 months at −20 °C

DNA molecular size marker

• Commercially available; the 50 bp ladder DNA marker gives a useful fragment range and eases sizing of the PCR amplicons

Reconditioning Solution for Dynabeads-oligo(dT)25

• 0.1 M NaOH (ACS grade or better)
• To prepare 50 mL of 1 M NaOH stock: weigh 2.0 g NaOH and complete to 50 mL with sterile distilled water (DNase/RNase-free) in a 50 mL sterile centrifugation tube; filter-sterilize through a 0.22 µm filter unit
• Make a 1:10 dilution in distilled water (DNase/RNase-free) for the 0.1 M NaOH working solution
• Stable for 6 months at room temperature (23 °C ± 3 °C)

Storage Buffer for Dynabeads-oligo(dT)25

• 250 mM Tris-HCl, pH 7.5 (molecular biology grade, commercially available)
• 20 mM EDTA (molecular biology grade, commercially available)
• 0.1% Tween 20 (molecular biology grade, commercially available)
• 0.02% sodium azide (NaN₃) (ACS grade)
• To prepare 50 mL from 1.0 M Tris-HCl, 0.5 M EDTA, Tween 20 solution, sodium azide powder and sterile distilled water (DNase/RNase-free):
• 12.5 mL of Tris-HCl (the source prints “12.5 mL of 0.1 M Tris-HCl” though the stocks named are 1.0 M Tris-HCl)
• 2.0 mL of 0.5 M EDTA
• 50 µL of Tween 20
• 0.01 g of sodium azide (NaN₃)
• 33.55 mL of sterile distilled water (DNase/RNase-free)
• Mix all reagents in a sterile 50 mL centrifugation tube and aliquot into smaller volumes for storing; stable for 6 months at room temperature (23 °C ± 3 °C)

9. Limitations

• This method does not differentiate between infectious and non-infectious viruses

10. Precautions

• Carry out virus extraction and concentration from green onions under a biological containment hood with a mask, sleeve protectors and double nitrile gloves
• TRIzol reagent contains phenol — handle carefully and dispose of as hazardous waste
• Ethidium bromide is a potent mutagen — wear nitrile gloves when handling it
• Ultraviolet light can cause eye damage — wear safety goggles
• Prepare all reagents in a laminar flow cabinet never exposed to noroviruses, norovirus PCR products, hepatitis A virus or HAV PCR products
• The laboratory supervisor must ensure the analysis is performed under ISO/IEC 17025:2005 (or the latest version) — general requirements for the competence of testing and calibration laboratories
• Where the method recommends 37 °C, the water bath may be at 37 °C ± 1 °C; for all other temperatures, ± 2 °C

References

• OPFLP-03, Concentration of Norovirus Genogroup II and Hepatitis A Virus from Contaminated Green Onions and Their Detection Using the Reverse-Transcriptase Polymerase Chain Reaction, July 2007, Health Products and Food Branch, Ottawa
• Kageyama, T., Kojima, S., Shinohara, M., Uchida, K., Fukushi, S., Hoshino, F. B., Takeda, N., and Katayama, K. 2003. Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. Journal of Clinical Microbiology, 41(4):1548–1557
• Anderson, A. D., et al. 2003. A waterborne outbreak of Norwalk-like virus among snowmobilers — Wyoming, 2001. Journal of Infectious Diseases, 187:303–306
• Guévremont, E., Brassard, J., Houde, A., Simard, C., and Trottier, Y.-L. 2006. Development of an extraction and concentration procedure and comparison of RT-PCR primer systems for the detection of hepatitis A virus and norovirus GII in green onions. Journal of Virological Methods, 134(1–2):130–135
• QIAGEN OneStep RT-PCR Kit Handbook, May 2002 (pp. 10–12)
• Invitrogen TRIzol product insert (15596-026)
• Dynal Biotech insert for Dynabeads-oligo(dT)25 (printed 0501, Rev. 004)
• Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 6.15