SOP: Detection of Norovirus Genogroups I and II Using the Conventional and Real-Time Reverse-Transcriptase Polymerase Chain Reaction

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

Total RNA is extracted from a food, clinical or environmental sample using an appropriate procedure — or from a cell-free sample (for instance, a norovirus suspension prepared from human stool samples) — with the QIAamp viral RNA extraction kit. The final RNA extract is used for conventional and/or real-time reverse-transcriptase polymerase chain reaction (RT-PCR), which amplifies a specific fragment depending on the primers used. The amplified human norovirus fragments are confirmed by sequencing after gel electrophoresis (conventional RT-PCR), or by the fluorescence reading of the TaqMan probe degraded by the Taq polymerase exonuclease activity during amplification (real-time RT-PCR). This protocol is routinely used to detect norovirus genogroup I (GI) and genogroup II (GII) RNA from oysters and green onions (see OPFLP-01 and OPFLP-03, the GIFSQ SOPs for those methods).

2. Apparatus required

• Thermal cycler (Eppendorf Mastercycler Gradient or equivalent)
• Spectrofluorometric thermal cycler (Stratagene Mx series or equivalent)
• Microwave oven or hot plate
• Submarine gel casting tray and buffer reservoir, power pack and appropriate 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, with photographic filters for ethidium bromide and/or SYBR Safe DNA gel stain
• Adjustable micropipettors covering 0.5–10 µL, 10–100 µL and 100–1000 µL, with filtered pipette tips
• Microcentrifuge for 1.5/2.0 mL microfuge tubes, speed range up to 20000 × g (Labnet International Spectrafuge 16 M or equivalent)
• Standard heating blocks (VWR scientific products or equivalent) or water bath for 1.5 mL microfuge tubes, maintaining 37–50°C (maintain 37°C ± 1°C where 37°C is recommended; ± 2°C for all other temperatures)
• Vortex mixer
• Sterile microfuge tubes (2.0 mL and 1.5 mL) and/or 96-well plate
• Tubes for PCR — thin-wall 0.2 mL or 0.5 mL capacity, strip or 96-well plate (depending on the thermal cycler model)
• Timer
• Container for ice or cooling rack
• Freezers capable of maintaining −20°C and −70°C
• Electroporation system (Bio-Rad Gene Pulser or equivalent); electroporation cuvettes, 0.1 cm gap (Bio-Rad 165-2089 or equivalent)
• Spectrophotometer (Thermo Scientific NanoDrop 1000 or equivalent)

3. Reagents and kits

• QIAamp viral RNA extraction kit (Qiagen 52904 or 52906, or equivalent)
• Qiagen OneStep RT-PCR kit (Qiagen 210210 or 210212, or equivalent)
• Stratagene Brilliant II QRT-PCR Core Reagent kit, 1-Step (Stratagene 600810 or equivalent)
• QIAquick gel extraction kit (Qiagen 28704 or 28706, or equivalent)
• TOPO TA Cloning kit (with pCR2.1-TOPO vector) with One Shot TOP10 Electrocomp E. coli (Invitrogen K4560-01 or K4560-40, or equivalent)
• NucleoSpin Plasmid kit (Macherey Nagel MCN540788050 or MCN540788250, or equivalent)
• Non-denatured ethanol, 96–99% (room temperature, 23°C ± 3°C)
• Kageyama primers for conventional RT-PCR: COG1F and COG1R (NoV GI); COG2F and COG2R (NoV GII) — see section 6
• Monroe primers for conventional RT-PCR and molecular characterization: MON-431, MON-432, MON-433 and MON-434 (NoV GI and GII) — see section 6
• Kageyama primers for real-time RT-PCR: COG1F and COG1R, with TaqMan probes RING1(a) and RING1(b) (NoV GI); COG2F and COG2R, with TaqMan probe RING2 (NoV GII) — see section 6
• X-GAL (5-Bromo-4-Chloro-3-Indolyl-β-D-Galactopyranoside) (BioShop XGA001 or equivalent)
• Kanamycin, 10 mg/mL (Invitrogen 18161-054 or equivalent)
• LB broth Miller (BioShop LBL407-50 or equivalent)
• EcoRI restriction endonuclease and buffer (Invitrogen 15202-021, or equivalent)
• BamHI restriction endonuclease and buffer (Invitrogen 15201-031, or equivalent)
• Phosphate-buffered saline (PBS), pH 7.4, 10× liquid (Gibco 70011-044 or equivalent)
• Dimethylformamide (DMF), reagent grade (BioShop DMF451 or equivalent)
• Agar (laboratory grade)
• 15 mL snap-cap tubes (e.g., Falcon)
• Agarose (molecular biology grade)
• DNA ladder, 100 bp (or equivalent)
• Ethidium bromide (molecular biology grade), or SYBR Safe DNA gel stain (Invitrogen S33102 or equivalent)
• Nucleic acid sample loading buffer, 10× (BlueJuice Gel Loading Buffer, Invitrogen 10816-015 or equivalent)
• RNase inhibitor (RNaseOUT Recombinant Ribonuclease Inhibitor, 40 U/µL, Invitrogen 10777-019 or equivalent)
• 0.5× TBE (Tris-Borate-EDTA) or 1× TAE (Tris-Acetate-EDTA) buffer
• RNase-free water (molecular biology grade)
• Salmon sperm DNA solution, 10 mg/mL (Invitrogen 15632-011 or equivalent)

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. Sample collection

• Determine the number of samples on the basis of the client’s needs (e.g., data collection or surveys) or investigational purposes (e.g., outbreaks)
• A clarified norovirus suspension previously prepared from human stool samples may also be used — aliquoted in 2 mL cryogenic vials and stored at −70°C

5. Procedure

Preparation of norovirus suspensions from human stool samples

Dilute stool samples in PBS (pH 7.4) to obtain 20% (w/v) suspensions

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Pulse-vortex vigorously until obtaining a homogeneous suspension

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Centrifuge the samples at 2000 × g for 3 min

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Transfer the supernatants to clean tubes

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Pulse-vortex vigorously until obtaining a homogeneous suspension

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Discard the previously used collection tube

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Centrifuge the samples at 16000 × g for 5 min

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Transfer the supernatants to clean tubes

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Discard the previously used collection tube

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Aliquot the clarified human stool suspensions in 2 mL cryogenic vials — extract viral RNA directly from the clarified suspensions or store at −70°C until needed

Viral RNA extraction from clarified stool suspensions (QIAamp Viral RNA Mini kit)

Ensure buffer AVL is completely dissolved before use; prepare buffer AVL–carrier RNA fresh (stable at 2–8°C for up to 48 h). The latest manufacturer’s instructions in the QIAamp Viral RNA Mini kit manual prevail over these instructions

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Pipet 560 µL of prepared buffer AVL containing carrier RNA into a 1.5 mL microfuge tube

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Add 140 µL of each sample to the tube containing buffer AVL–carrier RNA (buffers AVL and AW1 contain chaotropic salt, an irritant — wear gloves; they are not compatible with disinfecting agents that contain bleach)

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Pulse-vortex the microfuge tubes for 15 s

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Incubate the samples for 10 min at room temperature (23°C ± 3°C)

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Briefly centrifuge the microfuge tubes to remove drops from the inside of the lid (each centrifugation step is carried out at room temperature, 23°C ± 3°C)

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Add 560 µL of non-denatured ethanol (96–99%) to the samples

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Pulse-vortex the microfuge tubes for 15 s

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Transfer 630 µL of the solution to a QIAamp Mini spin column (in a 2 mL collection tube) without wetting the rim

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Close the caps and centrifuge the spin columns at 6000 × g for 1 min

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

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Transfer the remaining 630 µL of each solution to its respective spin column; centrifuge at 6000 × g for 1 min

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Repeat the transfer to a clean collection tube and discard the used one

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Add 500 µL of buffer AW1 to each spin column and close the lids; centrifuge at 6000 × g for 1 min

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

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Add 500 µL of buffer AW2 to each spin column and close the lids (buffers AW2 and AVE contain sodium azide as preservative, which is highly toxic — wear gloves)

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Centrifuge at 20000 × g for 3 min

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

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Add 60 µL of buffer AVE to each spin column (alternatively, a double elution with 2 × 30 µL of buffer AVE can be performed — two separate applications, each followed by centrifugation at 6000 × g for 1 min — see the QIAamp Viral RNA Mini Handbook)

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Close the caps and incubate at room temperature (23°C ± 3°C) for 1 min

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Centrifuge at 6000 × g 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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Add 20 units of RNase inhibitor (0.5 µL of RNaseOUT 40 U/µL) to the extracted RNA (take great care to avoid contact with RNases — they are very stable and difficult to inactivate; wear gloves while handling reagents and samples; use sterile, disposable RNase-free plasticware; bake glassware above 240°C for 4 h or more)

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Use the RNA directly in conventional or real-time RT-PCR, or store at −70°C until needed

Handling of sample units

Keep the RNA extracted from sample units frozen (−70°C) during storage and transport

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Keep the extracted RNA on ice while preparing the conventional or the real-time RT-PCR reactions

Preparation for analysis

Ensure that all reagents needed are available

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Prepare and verify the suitability of all controls — include a negative and a positive amplification control for both RT-PCR tests (conventional or real-time), specific to the primers used (the negative amplification control uses water as template, to ensure no contamination of the PCR mix; the positive amplification control uses purified NoV GI and/or GII RNA previously confirmed as positive in other experiments, or the corresponding cDNA clone from section 7, to ensure the PCR reagents and primers have the intended specificity)

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To estimate the norovirus titre by conventional RT-PCR (expressed in RT-PCR units): thaw a 2 mL cryogenic vial containing NoV GI and/or GII RNA on ice; make a 10-fold dilution series by adding 100 µL of virus stock to 900 µL of RNase-free water in a 1.5 mL microfuge tube (the 10⁻¹ dilution); repeat this step with 100 µL of the 10⁻¹ viral dilution in 900 µL of RNase-free water (the 10⁻² dilution); repeat to reach the 10⁻⁸ dilution

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For the real-time RT-PCR standard curve: generate, in triplicate, 10-fold serial dilutions (10⁸ to 10⁰ genomic equivalents) of purified linear cDNA plasmid (section 7) containing the PCR product obtained with each primer-set system, in a 5 ng/mL salmon sperm DNA solution — add 10 µL of plasmid stock to 90 µL of RNase-free water containing salmon sperm DNA (5 ng/mL) in a 1.5 mL microfuge tube (the 10⁻¹ dilution); repeat this step with 10 µL of the 10⁻¹ cDNA plasmid dilution in 90 µL of the same water (the 10⁻² dilution); repeat to reach the 10⁻⁸ dilution

Conventional RT-PCR for the amplification of norovirus RNA

Amplify RNA from each extracted RNA sample with the norovirus primer sets (Kageyama GI COG1F/COG1R and/or GII COG2F/COG2R; and/or Monroe MON-431/MON-432/MON-433/MON-434) using the Qiagen OneStep RT-PCR kit

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Add 5.0 µL of each extracted RNA sample to 20.0 µL of RT-PCR reaction mixture (section 6)

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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 NoV RNA to 20.0 µL of the same reaction mixture

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Insert PCR tubes or 96-well plate in a thermal cycler and run the RT-PCR amplification program in section 6

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

Agarose gel electrophoresis

Prepare a 3.0% (w/v) agarose gel in 0.5× TBE (Tris-Borate-EDTA) or 1× TAE (Tris-Acetate-EDTA) buffer — dissolve the agarose by stirring on a hot plate or microwaving for 1–2 min at high power until completely dissolved (a clear liquid with no particles in suspension)

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Cool the agarose to around 45°C; add concentrated ethidium bromide (EtBr) solution to obtain a final concentration of 0.5 µg/mL in the agarose gel (the source prints “0.5 g/ml”); mix gently while avoiding bubble formation (alternatively, SYBR Safe DNA gel stain can be used instead of EtBr — dilute the concentrated stain to a final 1:10000 in the agarose gel, see the SYBR Safe DNA gel stain product information; the addition of EtBr/SYBR Safe stain to the gel is optional if the gel is submerged in EtBr or SYBR Safe stain solution after migration; select the appropriate photographic filter for the stain used; EtBr is a potent mutagen — use nitrile gloves when handling)

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Pour into a gel tray; avoid bubble formation or bubble trapping; add a well-forming comb and allow the gel to solidify for about 20–30 min

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Prepare samples for electrophoresis: in clean microfuge tubes, mix 1.2 µL of tracking dye (nucleic acid loading buffer, 10× concentrated) with 10 µL of RT-PCR product

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When the agarose gel has solidified, remove the comb and place the tray with the gel in the electrophoresis apparatus; fill the reservoir with 0.5× TBE or 1× TAE buffer to cover the gel with buffer to a depth of 4 mm; gently pipet the samples (about 11.2 µL) into the wells of the submerged gel; include a DNA molecular-size marker (e.g., 100 bp DNA ladder) as well as positive, negative and reagent controls

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Connect the apparatus to the power supply with the cathode (−, black) at the top (near the sample wells) and the anode (+, red) at the bottom (the far end of the gel); apply about 100 V to the gel and run for about 30 min, or until the tracking dye has spread about two-thirds the length of the gel (the voltage and migration time can be modified to the size of the electrophoresis device and the length of the gel)

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Remove the gel from the tray and visualize the DNA bands by exposure to ultraviolet light (shortwave) using a transilluminator; photograph the gel (Polaroid 667 film or a digital processing system) for the record

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If EtBr/SYBR Safe was not added directly to the gel, remove the gel from the tray and stain the DNA by placing the gel in ethidium bromide solution (10 µg/mL — the source prints “10 g/ml”) or SYBR Safe 1× solution for 15 min; remove the gel with a gel scoop; rinse briefly with tap water; visualize the DNA bands by exposure to UV light (UV light can cause eye damage — wear safety goggles)

Reading conventional RT-PCR results

The amplicons generated by the NoV Kageyama GI (COG1F/COG1R), Kageyama GII (COG2F/COG2R) and Monroe (MON-431/MON-432/MON-433/MON-434) primers are double-stranded DNA fragments of 85 bp, 98 bp and 213 bp, respectively — a positive PCR test yields a DNA fragment specific to the targeted gene sequence and appears as an intense DNA band on an EtBr/SYBR Safe stained agarose gel; verify the molecular size of the band against a DNA molecular-size marker (e.g., 100 bp DNA ladder) run on the same gel

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A negative PCR test normally produces no visible DNA bands; in the extremely rare occurrence of bands not corresponding to the expected amplicon (non-specific amplification products), the sample is considered negative

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A specific band should appear for the targeted positive control — absence of a positive control band invalidates the test and the samples are re-amplified

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Any band corresponding to the positive control occurring in the negative control with the NoV primers indicates possible contamination of the RT-PCR reaction mixture — the whole batch is considered suspect and is discarded; the samples are re-amplified using a new reagent batch

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Any test sample showing a distinct band with the NoV primers, corresponding to its positive control, is a PRESUMPTIVE POSITIVE — since non-specific amplifications of the appropriate size have been observed occasionally with field (food, clinical or environmental) samples using Kageyama and Monroe primers in conventional RT-PCR, the products must be confirmed as a norovirus strain by sequencing (for food samples, send the extracted RNA obtained from positive samples to the Bureau of Microbial Hazards of Health Canada for further molecular characterization of the NoV strain)

Calculating RT-PCR units

After reading the RT-PCR results, determine the highest norovirus dilution showing a positive result — the end-point dilution

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Take the reciprocal of this dilution to give RT-PCR units per quantity used for the RT-PCR (i.e., 5.0 µL)

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To obtain a concentration per mL, multiply the concentration just obtained by a factor giving 1000 µL, i.e. 1 mL:

RT-PCR units per mL = (1 ÷ end-point dilution) × [1000 ÷ (µL of RNA used in the RT-PCR)]

• Worked example (from the source method): 5.0 µL of RNA used for the RT-PCR, and 10⁻⁶ the highest dilution in which a band was observed → 10⁶ × 200 = 2.0 × 10⁸ RT-PCR units per mL
• Where:
• end-point dilution = the highest norovirus dilution showing a positive result
• µL of RNA used in the RT-PCR = 5.0 in the worked example

Note: viral RNA titres are usually expressed as RT-PCR units per mL.

Real-time RT-PCR for the amplification of norovirus RNA

Amplify RNA from each extracted RNA sample in duplicate with the Kageyama primer and TaqMan probe sets (GI: COG1F, COG1R, RING1(a), RING1(b); GII: COG2F, COG2R, RING2 — see section 6) using the Stratagene Brilliant II QRT-PCR Core Reagent kit, 1-Step (or equivalent); perform, in triplicate, a minimum of 5 to 6 points of the corresponding validated standard curve of known copy number

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Add 2.0 µL of each extracted RNA sample, or clone used for the standard curve, to 23.0 µL of real-time RT-PCR reaction mixture (section 6)

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Set up reaction controls in triplicate — No Template Control (NTC): add 2.0 µL of RNase-free water to 23.0 µL of the same real-time RT-PCR reaction mixture; positive control: add 2.0 µL of HAV RNA previously confirmed as positive in other experiments, or cDNA from the corresponding clone, to 23.0 µL of the same mixture; in a two-step RT-PCR reaction a no-RT control could be included by omitting the reverse transcriptase enzyme — the no-RT control is expected to generate no real-time PCR signal with the specific primers and probe on previously confirmed NoV genomic RNA samples

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Insert PCR strip tubes or 96-well plate in the spectrofluorometric thermal cycler and run the RT-PCR amplification program in section 6 — take fluorescence readings at the annealing/extension temperature (60°C)

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When the real-time RT-PCR run is completed, analyse the reactions using the spectrofluorometric thermal cycler’s software; if necessary, the amplicons can be stored at 4°C for further analysis

Reading real-time RT-PCR results

Results are displayed in an amplification plot reflecting the change in fluorescence during cycling — a positive real-time RT-PCR test generates a Ct (threshold cycle), the cycle at which the fluorescence is determined to be statistically significant above the background signal of the fluorescence-labelled oligonucleotide within the PCR reaction; check for any abnormalities in the amplification plot

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If a standard curve is used, the expected concentration range of the sample should fall within the concentration range of the standard curve — Ct values would ideally fall between 15 and 35; a plot of Ct vs the logarithm of the copy number gives a straight line; an efficient real-time PCR assay has a slope of −3.3, an intercept of around 38 and an R² greater than 0.98; the amplification efficiency should be between 90 and 110% (optimal standard curves are based on amplification efficiencies as close to 100% as possible)

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A negative real-time RT-PCR test normally produces no fluorescence over the background signal of the probe for the entire run — it is validated with the positive control

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At a specific previously validated Ct, the fluorescence from the positive control should increase over the background fluorescence of the linear probe quench — absence of fluorescence in the positive control invalidates the test and the samples are re-amplified

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Any Ct occurring in the negative control with the NoV primers and probe set indicates possible contamination of the real-time RT-PCR reaction mixture — the whole batch is considered suspect and is discarded; the samples are re-amplified using a new reagent batch

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Any sample showing a Ct with the NoV primers and probe set is considered positive when the NTCs are negative, or when the NTC Ct is at least 5 cycles from the highest Ct value of the samples analysed (for food samples, send the extracted RNA obtained from positive samples to the Bureau of Microbial Hazards of Health Canada for further molecular characterization of the NoV strain)

Calculating genomic equivalent units

The unknown sample’s Ct value must fall on the standard curve to be quantified in genomic equivalents/volume — estimate the initial genomic equivalent values of the cDNA from the mean of the duplicate threshold cycle (Ct) values of the sample analysed, compared against its respective (NoV GI or GII) standard curve

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To obtain the initial concentration in genomic equivalents per µL, divide the concentration obtained above by the volume of RNA used as template:

Genomic equivalents per µL = (genomic equivalents from the standard curve) ÷ (µL of RNA used as template)

• Worked example (from the source method): 2.0 µL of RNA used for the real-time RT-PCR, and the mean Ct value corresponds to 4.0 × 10⁶ genomic equivalents on the standard curve → 4.0 × 10⁶ ÷ 2 = 2.0 × 10⁶ genomic equivalents per µL
• Where:
• genomic equivalents from the standard curve = value read from the standard curve at the sample’s mean Ct
• µL of RNA used as template = 2.0 in the worked example

Note: viral RNA concentrations are usually expressed as genomic equivalents per µL of RNA used as template; with appropriate calculations this can be expressed as genomic equivalents per unit of sample tested.

6. Primer sequences, temperature cycling programs and reaction mixtures

Conventional RT-PCR primers

Kageyama primers (Kageyama et al. 2003 — see References) for the detection of NoV GI (85 bp fragment):

• COG1F (forward, 20 bases): 5′-CGY TGG ATG CGI TTY CAT GA-3′
• COG1R (reverse, 22 bases): 5′-CTT AGA CGC CAT CAT CAT TYA C-3′

Kageyama primers for the detection of NoV GII (98 bp fragment):

• COG2F (forward, 26 bases): 5′-CAR GAR BCI ATG TTY AGR TGG ATG AG-3′
• COG2R (reverse, 21 bases): 5′-TCG ACG CCA TCT TCA TTC ACA-3′

Monroe primers (Anderson et al. 2003 — see References) for the detection and molecular characterization of NoV GI and GII (213 bp fragment):

• MON431 (forward, 20 bases): 5′-TGG ACI AGR GGI CCY AAY CA-3′
• MON432 (forward, 20 bases): 5′-TGG ACI CGY GGI CCY AAY CA-3′
• MON433 (reverse, 21 bases): 5′-GAA YCT CAT CCA YCT GAA CAT-3′
• MON434 (reverse, 21 bases): 5′-GAA SCG CAT CCA RCG GAA CAT-3′

Standard 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 (I) = A, C, G, T; X = A, C, G, T.

Note: synthesis of oligonucleotide primers can usually be contracted out to a local university; alternatively, many biotechnology firms offer a custom synthesis service; for better results, use primers purified by High Pressure/Performance Liquid Chromatography (HPLC).

Temperature cycling program — conventional RT-PCR

Using the Kageyama NoV GI and GII primer systems, 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 (HotStarTaq DNA Polymerase is activated; Omniscript and Sensiscript Reverse Transcriptases are inactivated; the cDNA template is denatured)
• Step 3 — 40 cycles of: denaturation 30 s at 94°C; annealing 30 s at 52°C; extension 45 s at 72°C
• Step 4 — Final elongation: 5 min at 72°C

Using the Monroe NoV GI/GII primer system, 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 (HotStarTaq DNA Polymerase is activated; Omniscript and Sensiscript Reverse Transcriptases are inactivated; the cDNA template is denatured)
• Step 3 — 40 cycles of: denaturation 30 s at 94°C; annealing 30 s at 52°C; extension 45 s at 72°C
• Step 4 — Final elongation: 5 min at 72°C

Note: the use of thermal cyclers other than the models stated above, or of another RT-PCR reagent kit than the Qiagen OneStep RT-PCR kit, may alter the performance of the RT-PCR reactions — it may be necessary to optimize the cycling parameters for different models or reagent kits (see the Qiagen OneStep RT-PCR Kit Handbook).

Qiagen OneStep RT-PCR mix (batch for 20 reactions; all stock solutions stored at −20°C until used)

Kageyama NoV GI or GII system — batch / per tube / final concentration (final concentrations are per 25 µL of reaction: 20.0 µL of mix + 5.0 µL of template):

• RNase-free water: 200.0 µL / 10.0 µL / —
• 5× Qiagen OneStep RT-PCR buffer: 100.0 µL / 5.0 µL / 1×
• dNTP mix (10 mM of each dNTP): 20.0 µL / 1.0 µL / 400 µM
• COG1F or COG2F primer (10 µM): 30.0 µL / 1.5 µL / 0.6 µM
• COG1R or COG2R primer (10 µM): 30.0 µL / 1.5 µL / 0.6 µM
• Qiagen OneStep RT-PCR enzyme mix: 20.0 µL / 1.0 µL / —
• Total volume: 400.0 µL batch / 20.0 µL per tube — distribute 20.0 µL per tube; template per tube: 5.0 µL

Monroe NoV GI/GII system — batch / per tube / final concentration (per 25 µL of reaction):

• RNase-free water: 140.0 µL / 7.0 µL / —
• 5× Qiagen OneStep RT-PCR buffer: 100.0 µL / 5.0 µL / 1×
• dNTP mix (10 mM of each dNTP): 20.0 µL / 1.0 µL / 400 µM
• MON431 primer (10 µM): 30.0 µL / 1.5 µL / 0.6 µM
• MON432 primer (10 µM): 30.0 µL / 1.5 µL / 0.6 µM
• MON433 primer (10 µM): 30.0 µL / 1.5 µL / 0.6 µM
• MON434 primer (10 µM): 30.0 µL / 1.5 µL / 0.6 µM
• Qiagen OneStep RT-PCR enzyme mix: 20.0 µL / 1.0 µL / —
• Total volume: 400.0 µL batch / 20.0 µL per tube — distribute 20.0 µL per tube; template per tube: 5.0 µL

Note: the RT-PCR protocol follows the manufacturer’s instructions in the Qiagen OneStep RT-PCR kit manual — always follow the latest manufacturer’s instructions, which prevail over these instructions; take great care to avoid contact with ubiquitous RNases; always store the extracted RNA with 20 units of RNase inhibitor and wear gloves; use sterile, disposable RNase-free plasticware; bake glassware above 240°C for 4 h or more; all reagents, DNase/RNase-free water, pipette tips and other materials contacting samples or RT-PCR reagents should be sterile or autoclaved; to avoid contamination, prepare all reagents in a laminar-flow cabinet that has never been exposed to NoV or NoV RT-PCR products; to avoid non-specific amplification, keep all reagents on ice or on a refrigerated rack while preparing the mix; 25 µL of RT-PCR mix is used in 200 µL tubes.

Real-time RT-PCR primers and TaqMan probes

Kageyama primers (Kageyama et al. 2003 — see References) for the detection of NoV GI (85 bp fragment) with a TaqMan assay:

• COG1F (forward, 20 bases): 5′-CGY TGG ATG CGI TTY CAT GA-3′
• COG1R (reverse, 22 bases): 5′-CTT AGA CGC CAT CAT CAT TYA C-3′
• RING1(a) probe (20 bases): 5′-AGA TYG CGA TCY CCT GTC CA-3′
• RING1(b) probe (20 bases): 5′-AGA TCG CGG TCT CCT GTC CA-3′

Kageyama primers for the detection of NoV GII (98 bp fragment) with a TaqMan assay:

• COG2F (forward, 26 bases): 5′-CAR GAR BCI ATG TTY AGR TGG ATG AG-3′
• COG2R (reverse, 21 bases): 5′-TCG ACG CCA TCT TCA TTC ACA-3′
• RING2 probe (30 bases): 5′-TGG GAG GGC GAT CGC AAT CT-3′

The RING1(a), RING1(b) and RING2 TaqMan probes were ordered as 5′HEX (Hexachlorofluorescein)–3′IBQ (Iowa Black Quencher); other fluorochrome/quencher combinations are possible. The same degenerate-base codes apply as for the conventional primers (above).

Note: synthesis of oligonucleotide primers and TaqMan probes can usually be contracted out to a local university; alternatively, many biotechnology firms offer a custom synthesis service; order oligonucleotides purified by High Pressure/Performance Liquid Chromatography (HPLC) for real-time RT-PCR applications.

Temperature cycling program — real-time RT-PCR

Using the Kageyama NoV GI primer and probe system, set the spectrofluorometric thermal cycler for the following sequence of cycling parameters:

• Step 1 — Reverse transcription: 30 min at 50°C
• Step 2 — Initial PCR activation: 10 min at 95°C (SureStart Taq DNA Polymerase is activated; the reverse transcriptase is inactivated; the cDNA template is denatured)
• Step 3 — 45 cycles of: denaturation 15 s at 95°C; annealing + extension 60 s at 60°C

Using the Kageyama NoV GII primer and probe system, set the spectrofluorometric thermal cycler for the following sequence of cycling parameters:

• Step 1 — Reverse transcription: 30 min at 50°C
• Step 2 — Initial PCR activation: 10 min at 95°C (SureStart Taq DNA Polymerase is activated; the StrataScript reverse transcriptase is inactivated; the cDNA template is denatured)
• Step 3 — 45 cycles of: denaturation 15 s at 95°C; annealing + extension 60 s at 60°C

Note: the use of spectrofluorometric thermal cyclers other than the models stated above, or of another QRT-PCR reagent kit than the Stratagene Brilliant II QRT-PCR Core Reagent kit 1-Step, may alter the performance of the QRT-PCR reactions — it may be necessary to optimize the cycling parameters, or the primer, probe or MgCl₂ concentrations, for different models or kits (see the Brilliant II QRT-PCR Core Reagent Kit, 1-Step Instruction Manual).

Stratagene Brilliant II QRT-PCR Core Reagent mix, 1-Step (batch for 102 reactions, for a 96-well plate; all stock solutions stored at −20°C until used)

Kageyama NoV GI system — batch / per tube / final concentration:

• RNase-free water: 1362.25 µL / 12.375 µL / — (the batch components as printed sum to 2446.0 µL against the printed batch total of 2346.0 µL; the per-tube volumes sum to the printed 23.0 µL per tube)
• 10× core RT-PCR buffer: 255.0 µL / 2.5 µL / 1×
• Magnesium chloride (50 mM): 255.0 µL / 2.5 µL / 5.0 mM
• COG1F primer (10 µM): 102.0 µL / 1.0 µL / 0.4 µM
• COG1R primer (10 µM): 102.0 µL / 1.0 µL / 0.4 µM
• RING1(a) probe (10 µM): 76.5 µL / 0.75 µL / 0.3 µM
• RING1(b) probe (10 µM): 25.5 µL / 0.25 µL / 0.1 µM
• dNTP mix (20 mM, 5 mM of each dNTP): 102.0 µL / 1.0 µL / 800 µM
• Reverse transcriptase: 102.0 µL / 1.0 µL / —
• Reference dye (ROX), 1 mM, diluted 1:500 in nuclease-free PCR-grade water: 38.25 µL / 0.375 µL / 0.03 µM
• SureStart Taq DNA polymerase (5 U/µL): 25.5 µL / 0.25 µL / 0.05 U/µL
• Total volume: 2346.0 µL batch / 23.0 µL per tube — distribute 23.0 µL per tube; template per tube: 2.0 µL

Kageyama NoV GII system — batch / per tube / final concentration:

• RNase-free water: 1338.75 µL / 13.125 µL / —
• 10× core RT-PCR buffer: 255.0 µL / 2.5 µL / 1×
• Magnesium chloride (50 mM): 255.0 µL / 2.5 µL / 5.0 mM
• COG2F primer (10 µM): 102.0 µL / 1.0 µL / 0.4 µM
• COG2R primer (10 µM): 102.0 µL / 1.0 µL / 0.4 µM
• RING2 probe (10 µM): 25.5 µL / 0.25 µL / 0.1 µM
• dNTP mix (20 mM, 5 mM of each dNTP): 102.0 µL / 1.0 µL / 800 µM
• Reverse transcriptase: 102.0 µL / 1.0 µL / —
• Reference dye (ROX), 1 mM, diluted 1:500 in nuclease-free PCR-grade water: 38.25 µL / 0.375 µL / 0.03 µM
• SureStart Taq DNA polymerase (5 U/µL): 25.5 µL / 0.25 µL / 0.05 U/µL
• Total volume: 2346.0 µL batch / 23.0 µL per tube — distribute 23.0 µL per tube; template per tube: 2.0 µL

Note: if ROX is omitted as a passive reference dye, replace it with an equivalent volume of RNase-free water; to compensate for non-PCR-related variations in fluorescence, some spectrofluorometric thermal cyclers require ROX as a passive reference dye; the real-time RT-PCR protocol follows the manufacturer’s instructions in the Brilliant II QRT-PCR Core Reagent kit 1-Step manual — always follow the latest manufacturer’s instructions, which prevail over these instructions; take great care to avoid contact with ubiquitous RNases; always store the extracted RNA with 20 units of RNase inhibitor and wear gloves; use sterile, disposable RNase-free plasticware; bake glassware above 240°C for 4 h or more; all reagents, DNase/RNase-free water, pipette tips and other materials contacting samples or RT-PCR reagents should be sterile or autoclaved; to avoid contamination, prepare all reagents in a laminar-flow cabinet that has never been exposed to NoV or NoV RT-PCR products; keep all reagents on ice or on a refrigerated rack while preparing the mix; centrifuge before PCR amplification.

7. Preparation of the cDNA clone for the real-time RT-PCR standard curve

Production of the cDNA fragment by conventional RT-PCR

Amplify NoV RNA with the real-time primer sets (GI: COG1F and COG1R; GII: COG2F and COG2R) using the Qiagen OneStep RT-PCR kit, with the conventional RT-PCR program in section 6

↓

Add 10.0 µL of NoV RNA sample to 40.0 µL of conventional RT-PCR reaction mixture (double the section 6 recipe)

↓

After the RT-PCR is completed, analyse the RT-PCR product by agarose gel electrophoresis (section 5); if necessary, the amplicons can be stored at 4°C

↓

Prepare the sample for electrophoresis: in a clean microfuge tube, mix 4.0 µL of tracking dye (nucleic acid loading buffer, 10× concentrated) and 40 µL of RT-PCR product — the amplicons generated by the NoV GI primers and GII primers are double-stranded DNA fragments of 85 bp and 98 bp, respectively

Purification of the RT-PCR product (QIAquick gel extraction kit)

All reagents used here are provided in the kit with the exception of ethanol 96–99% — add ethanol (96–99%) to Buffer PE before use. The latest manufacturer’s instructions in the QIAquick gel extraction kit manual prevail over these instructions

↓

Excise the DNA fragment from the agarose gel with a clean, sharp scalpel; minimize the size of the gel slice by removing extra agarose

↓

Weigh the gel slice in a colourless microtube; add 6 volumes of Buffer QG to 1 volume of gel (100 mg ≈ 100 µL)

↓

Incubate at 50°C for 10 min, or until the gel slice has completely dissolved — to help dissolve the gel, mix by vortexing the tube every 2–3 min during the incubation (for 3% gels it may be necessary to increase the incubation time)

↓

After the gel slice has dissolved completely, check that the colour of the mixture is yellow (similar to Buffer QG without dissolved agarose) — if the colour is orange or violet, add 10 µL of 3 M sodium acetate (pH 5.0) and mix; the mixture will turn yellow (adsorption of DNA to the QIAquick membrane is efficient only at pH ≤ 7.5; Buffer QG contains a pH indicator, yellow at pH ≤ 7.5 and orange or violet at higher pH)

↓

Add 1 gel volume of isopropanol to the sample and mix

↓

Place a QIAquick spin column in a provided 2 mL collection tube

↓

To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min at 17900 × g (13000 rpm) in a conventional table-top microcentrifuge at room temperature (23°C ± 3°C) — the maximum volume of the column reservoir is 800 µL; for sample volumes above 800 µL, load and spin again

↓

Discard the flow-through and place the QIAquick column back in the same collection tube (collection tubes are reused to reduce plastic waste)

↓

Add 0.5 mL of Buffer QG to the QIAquick column and centrifuge for 1 min at 17900 × g (13000 rpm)

↓

To wash, add 0.75 mL of Buffer PE to the QIAquick column and centrifuge for 1 min at 17900 × g (13000 rpm)

↓

Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 17900 × g (13000 rpm) (residual ethanol from Buffer PE is not completely removed unless the flow-through is discarded before this additional centrifugation)

↓

Place the QIAquick column into a clean 1.5 mL microcentrifuge tube

↓

To elute DNA, add 50 µL of Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the centre of the QIAquick membrane; let stand for 1 min; centrifuge the column for 1 min at 17900 × g (13000 rpm) (dispense the elution buffer directly onto the membrane for complete elution; the average eluate volume is 48 µL from 50 µL of elution buffer; maximum elution efficiency is achieved between pH 7.0 and 8.5 — when using water, keep the pH within this range and store DNA at −20°C, since DNA may degrade without a buffering agent; the purified DNA can also be eluted in TE buffer — 10 mM Tris·Cl, 1 mM EDTA, pH 8.0 — but the EDTA may inhibit subsequent enzymatic reactions)

↓

Verify the purified DNA by agarose gel electrophoresis (section 5), loading 9 µL of purified product and 1 µL of tracking dye (nucleic acid loading buffer, 10× concentrated)

Cloning reaction with the TOPO TA Cloning kit, and transformation of competent cells

All reagents used here are provided in the kit with the exception of LB medium and LB agar plates containing the appropriate antibiotic and X-Gal. The latest manufacturer’s instructions in the TOPO TA Cloning user manual prevail over these instructions

↓

Just before beginning, warm the vial of S.O.C medium to room temperature (23°C ± 3°C)

↓

Warm selective plates (LB plate containing 50 µg/mL of kanamycin) at 37°C for 30 min

↓

Spread 40 µL of 40 mg/mL X-Gal in dimethylformamide (DMF) on each LB agar plate and incubate at 37°C until ready for use

↓

Place the electroporation cuvettes on ice

↓

Thaw on ice 1 vial of One Shot TOP10 Electrocompetent E. coli cells

↓

Dilute the salt solution 4-fold by adding 15 µL of water to 5 µL of salt solution

↓

Set up the TOPO cloning reaction by adding 4 µL of purified PCR product to 1 µL of diluted salt solution and 1 µL of pCR2.1-TOPO vector

↓

Mix gently and incubate for 5 min at room temperature

↓

Place the reaction on ice

↓

Add 2 µL of the TOPO cloning reaction into a vial of One Shot TOP10 Electrocompetent E. coli cells and mix gently (do not mix by pipetting up and down)

↓

Carefully transfer the solution to a 0.1 cm cuvette, avoiding the formation of bubbles

↓

Electroporate the sample using your own protocol and electroporator

↓

Immediately add 250 µL of room-temperature S.O.C medium

↓

Transfer the solution to a 15 mL snap-cap tube (e.g., Falcon) and shake for at least 1 h at 37°C to allow expression of the antibiotic resistance genes

↓

Spread 10–50 µL from each transformation on a pre-warmed LB plate containing X-Gal and 50 µg/mL kanamycin, and incubate overnight at 37°C

↓

Take 10 white colonies and culture them overnight at 37°C in 5 mL of LB medium containing 50 µg/mL kanamycin

↓

Isolate plasmid DNA to confirm the presence of the corresponding NoV insert (transformation of E. coli can also be performed chemically — see the Invitrogen user manual)

Isolation of plasmid DNA from E. coli (NucleoSpin Plasmid kit)

All reagents used here are provided in the kit with the exception of ethanol 96–99% and 1.5 mL microcentrifuge tubes. The latest manufacturer’s instructions in the NucleoSpin Plasmid kits user manual prevail over these instructions

↓

Before beginning, add the entire contents of the provided vial of RNase A to the bottle of Buffer A1; mix well and store at 4°C for up to 6 months

↓

Add 96–99% non-denatured ethanol to Buffer A4 before use — the appropriate volume of ethanol is printed on the Buffer A4 bottle

↓

Pipet 1.5 mL of saturated E. coli culture and centrifuge at 11000 × g for 30 s to pellet the bacterial cells

↓

Discard the supernatant

↓

Resuspend the pellet in 250 µL of Buffer A1 by vigorous vortexing (check Buffer A2 for precipitated SDS before use — if a white precipitate is visible, warm the buffer for several minutes at 30–40°C until the precipitate is dissolved completely; cool the buffer to room temperature, 23°C ± 3°C)

↓

Add 250 µL of Buffer A2 to the suspension; mix gently by inverting the tube 6–8 times; incubate at room temperature for 5 min (do not vortex, to avoid the release of contaminating chromosomal DNA into the suspension)

↓

Add 300 µL of Buffer A3 to the suspension; mix gently by inverting the tube 6–8 times (buffers A3 and AW contain guanidine hydrochloride, which is highly toxic — wear gloves)

↓

Centrifuge the suspension at 11000 × g in a microcentrifuge for 5 min — repeat this step if the supernatant is not clear

↓

Place a NucleoSpin Plus column in a 2 mL collection tube and load the supernatant into the spin column

↓

Centrifuge at 11000 × g for 1 min and discard the flow-through

↓

Wash the NucleoSpin column with 500 µL of Buffer AW

↓

Centrifuge at 11000 × g for 1 min and discard the flow-through

↓

Reinsert the NucleoSpin column in the 2 mL collection tube and add 600 µL of Buffer A4 (with ethanol) to the NucleoSpin column

↓

Centrifuge at 11000 × g for 1 min and discard the flow-through

↓

Recentrifuge at 11000 × g for 2 min to remove residual ethanol from the silica membrane inside the spin column

↓

Place the NucleoSpin column in a 1.5 mL or 2 mL microcentrifuge tube (not included with the kit); elute the DNA by adding 50 µL of Buffer AE (5 mM Tris-HCl, pH 8.5) to the column; incubate 1 min at room temperature (23°C ± 3°C)

↓

Centrifuge at 11000 × g for 1 min — the plasmid DNA is now collected in Buffer AE at the bottom of the centrifuge tube

Confirmation of the presence of the insert

Add 2.0 µL of purified DNA plasmid, 2.0 µL of restriction endonuclease buffer (provided with the restriction endonuclease), 15.0 µL of RNase/DNase-free water and 1.0 µL of EcoRI (10 U/µL) restriction endonuclease in a microfuge tube

↓

Incubate overnight in a water bath or heating block at 37°C

↓

After the reaction is completed, prepare the sample for electrophoresis: in a clean microfuge tube, mix 10 µL of the digested plasmid product and 1 µL of tracking dye (nucleic acid loading buffer, 10× concentrated); analyse by agarose gel electrophoresis (section 5)

↓

The two DNA fragments generated by the EcoRI restriction endonuclease are 3921 bp (plasmid) and 95 bp (85 bp of NoV GI cDNA produced with COG1 primers + 10 bp of plasmid), or 3921 bp (plasmid) and 108 bp (98 bp of NoV GII cDNA produced with COG2 primers + 10 bp of plasmid) — bands of 95 bp or 108 bp against a DNA molecular-size marker (e.g., 100 bp DNA ladder) run on the same gel confirm NoV GI and/or NoV GII cDNA, respectively (it is advisable to sequence the insert to confirm its integrity — the construct may be sequenced using M13 forward and reverse primers; DNA sequencing can usually be contracted out to a local university or a biotechnology firm)

Plasmid linearization for use as a standard

Add 35.0 µL of purified DNA plasmid, 12.0 µL of restriction endonuclease buffer (provided with the restriction endonuclease), 65.0 µL of RNase/DNase-free water and 10.0 µL of BamHI (10 U/µL) restriction endonuclease in a microfuge tube

↓

Incubate 4 h in a water bath or heating block at 37°C

↓

Inactivate the restriction endonuclease by incubating 20 min in a water bath or heating block at 55°C

↓

After the reaction is completed, prepare the sample for electrophoresis: in a clean microfuge tube, mix 2.0 µL of the digested plasmid product, 8.0 µL of RNase/DNase-free water and 1 µL of tracking dye (nucleic acid loading buffer, 10× concentrated); analyse by agarose gel electrophoresis (section 5)

↓

The DNA fragment generated by the BamHI restriction endonuclease is 4016 bp (3931 bp plasmid + 85 bp NoV GI cDNA produced with COG1 primers) or 4029 bp (3931 bp plasmid + 98 bp NoV GII cDNA produced with COG2 primers) — only one band should appear on the agarose gel, confirming linearization of the cDNA clone

Standard curve preparation for real-time RT-PCR

Determine the concentration in g/µL of the linearized DNA plasmid stock (from the linearization above) with a spectrophotometer

↓

Evaluate the molar mass: bp length (plasmid + NoV insert) × 649 g/mol [molar mass for 1 kb]

• NoV GI: (3931 bp + 85 bp) × 649 g/mol = 2.606 × 10⁶ g/mol
• NoV GII: (3931 bp + 98 bp) × 649 g/mol = 2.615 × 10⁶ g/mol

↓

Evaluate the copy number (genomic equivalents) per µL:

Copy number per µL = [(concentration of linearized plasmid, g/µL) ÷ (molar mass)] × (6.023 × 10²³)

• Worked example (from the source method): for linearized plasmid containing NoV GII cDNA at 143.89 ng/µL, i.e. 1.439 × 10⁻⁷ g/µL:
  copy number per µL = [(1.439 × 10⁻⁷ g/µL) ÷ (2.615 × 10⁶ g/mol)] × (6.023 × 10²³) = 33.144 × 10⁹, or 3.314 × 10¹⁰
• Where:
• concentration of linearized plasmid = spectrophotometer reading, in g/µL
• molar mass = bp length (plasmid + NoV insert) × 649 g/mol
• 6.023 × 10²³ = Avogadro’s number

↓

Dilute the linear plasmid stock in RNase/DNase-free water to obtain a concentration of 1 × 10⁹ copies (genomic equivalents) per µL

↓

Generate the standard curves as in section 5 (the 10-fold dilution series in 5 ng/mL salmon sperm DNA)

8. Limitations

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

9. Precautions

• Take appropriate laboratory measures when handling human norovirus; Biosafety Level 2 practices and containment are required for activities with contaminated materials; for more information consult the Public Health Agency of Canada Level 2 biosafety guidelines
• Wear a laboratory coat; wear gloves when direct contact with infectious materials is unavoidable; wear sleeve protectors and (nitrile) gloves for work in the biosafety cabinet
• Buffers AVL and AW1 contain chaotropic salt, which is an irritant — wear gloves when handling; they are not compatible with disinfecting agents that contain bleach
• Buffers AW2 and AVE contain sodium azide as preservative, which is highly toxic — take appropriate safety measures and wear gloves when handling
• Buffers A3 and AW contain guanidine hydrochloride, which is highly toxic — take appropriate safety measures and wear gloves when handling
• Dispose of all solutions, buffers and reagents according to the waste-disposal guidelines
• Take great care to avoid contact with ubiquitous RNases — they are very stable and difficult to inactivate; always store the extracted RNA with 20 units of RNase inhibitor and wear gloves while handling reagents and samples; use sterile, disposable RNase-free plasticware; bake glassware above 240°C for 4 h or more before use
• Ethidium bromide is a potent mutagen — handle with nitrile gloves
• UV light can cause eye damage — wear safety goggles
• The laboratory supervisor must ensure that completion of the analysis is done in accordance with ISO/IEC 17025:2005 (or latest version): General requirements for the competence of testing and calibration laboratories

References

• Health Canada, Compendium of Analytical Methods, Volume 5 — Laboratory Procedure OPFLP-10, March 2010: “Detection of norovirus genogroups I and II using the conventional and real-time reverse-transcriptase polymerase chain reaction.”
• Anderson A.D. et al. 2003. A waterborne outbreak of Norwalk-like virus among snowmobilers — Wyoming 2001. Journal of Infectious Diseases, 187: 303–306.
• Brilliant II QRT-PCR Core Reagent Kit, 1-Step Instruction Manual. 2006.
• David S.T. et al. 2007. An outbreak of norovirus caused by consumption of oysters from geographically disperse harvest sites, British Columbia, Canada, 2004. Foodborne Pathogens and Disease, 4: 349–358.
• Guévremont E. et al. 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: 130–135.
• Houde A. et al. 2006. Comparative evaluation of RT-PCR, nucleic acid sequence based amplification (NASBA) and real-time RT-PCR for detection of noroviruses in faecal material. Journal of Virological Methods, 135: 163–172.
• Jothikumar N. et al. 2005. Rapid and sensitive detection of noroviruses by using TaqMan-based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples. Applied and Environmental Microbiology, 71: 1870–1875.
• Kageyama T. et al. 2003. Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. Journal of Clinical Microbiology, 41: 1548–1557.
• Loisy F. et al. 2005. Real-time RT-PCR for norovirus screening in shellfish. Journal of Virological Methods, 123: 1–7.
• NucleoSpin Plasmid kits user Manual. March 2008.
• Pang X.L. et al. 2005. Multiplex real time RT-PCR for the detection and quantitation of norovirus genogroups I and II in patients with acute gastroenteritis. Journal of Clinical Virology, 33: 168–171.
• QIAamp Viral RNA Mini Handbook. December 2007.
• Qiagen OneStep RT-PCR Kit Handbook. February 2008.
• QIAquick Spin Handbook. March 2008. pp. 25–26.
• SYBR Safe DNA gel stain. July 2006.
• TOPO TA Cloning user Manual. April 2006.
• Trujillo A.A. et al. 2006. Use of TaqMan real-time reverse transcription-PCR for rapid detection, quantification, and typing of noroviruses. Journal of Clinical Microbiology, 44: 1405–1412.