Concentration Of Norovirus Genogroup II and Hepatitis A Virus From Contaminated Green Onions and Their Detection Using The Reverse-transcriptase polymerase Chain Reaction

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OPFLP-03
July 2007

Laboratory Procedure

HEALTH PRODUCTS AND FOOD BRANCH

OTTAWA

1. Application

This method is applicable to the concentration and detection of noroviruses belonging to genogroup II and hepatitis A virus from contaminated fresh green onions. This method will not differentiate between infectious and non-infectious viruses.

2. Description

Initially, this method has shown to produce satisfactory results with artificially contaminated (spiked) fresh green onions with noroviruses genogroup II and hepatitis A virus.

3. Principle

The viral particles are eluted and concentrated by repeated pipetting/washing of the surface of fresh green onions with a pH 9.5 tryptose phosphate broth-glycine buffered solution. Concentration of viral load is performed by a polyethylene glycol (PEG) precipitation step. Viral RNAs are extracted and purified using a combination of TRIzol^®-chloroform and poly (dT) magnetic beads procedures. These magnetic beads bind the polyadenylated (poly-A tail) messenger RNA (mRNA) of noroviruses and hepatitis A virus. The final extract is then subjected to a reverse-transcriptase polymerase chain reaction (RT-PCR) procedure which amplifies a specific fragment depending on the primer sets used (i.e. norovirus genogroup II or hepatitis A virus).

4. Definition of Terms

4.1 See Appendix A of Volume 3.

5. Collection of Samples

5.1 The number of samples will have to be determined on the basis of the client needs (e.g. data collection or surveys) or investigational purposes (e.g. outbreaks).

6. Materials and Special Equipment

Note:
The Laboratory Supervisor must ensure that completion of the analysis, described in this method, must be done in accordance with the International Standard referred to as "ISO/IEC 17025:2005 (or latest version). General requirements for the competence of testing and calibration laboratories".

6.1 Thermal cycler (Eppendorf Mastercyler Gradient or equivalent).

6.2 Microwave oven or hot plate.

6.3 Submarine gel casting tray and buffer reservoir, power pack and an appropriate comb.

6.4 Shortwave UV light table (transilluminator) to visualize stained DNA in agarose gels.

6.5 Photo documentation system (optional, for photographic records), including Polaroid camera (hand-held or fixed), hood and Polaroid 667 film or equivalent.

6.6 Adjustable micropipettors: to cover range of volumes: 0.5 to 10 μl, 10 to 100 μL, and 100 to 1000 μL with specific sterile filtered pipet tips.

6.7 Standard heatblocks (VWR scientific products or equivalent) capable of accommodating 1.5 mL microfuge tubes and capable of maintaining a temperature of 65°C and 90°C.

6.8 Platform balance with a resolution of 0.01g.

Note:
It is the responsibility of each laboratory to ensure that the block heaters or water baths are maintained at the recommended temperatures. Where 37°C is recommended the waterbath may be at 37°C +/- 1°C. For all other temperatures it may be +/- 2°C.

6.9 Vortex mixer.

6.10 Microfuge tubes - 2.0 mL and 1.5 mL capacity.

6.11 SORVALL RC-5B Plus refrigerated centrifuge with rotors number SS-34 (8 X 50 mL Polypropylene Copolymer Oak Ridge tubes) or equivalent.

6.12 50 mL (Polypropylene Copolymer Oak Ridge centrifugation tubes or Polypropylene centrifugation tubes).

6.13 IEC DPR-6000 refrigerated centrifuge or equivalent.

6.14 IEC DPR-6000 rotors for 15 mL tubes or equivalent.

6.15 15 mL polypropylene centrifugation tubes (Sarstead product number 62.554.002 or better).

6.16 Dynal Magnetic bead attractors model MPC-S (product number 120.20, no substitution).

6.17 Dynal Biotech Sample Mixer (product number 947.01 or equivalent).

6.18 Sorvall Legend RT refrigerated centrifuge with Heraeus rotor number 3332.

6.19 Tubes for PCR - thin wall 0.2 mL or 0.5 mL capacity (depending on thermal cycler model).

6.20 Timer.

6.21 Container for ice.

6.22 Magnetic stirrer.

6.23 Freezers capable of maintaining - 20°C and - 70°C.

6.24 Qiagen OneStep RT-PCR Kit (product numbers 210210 or 210212, no substitution). (8.4)

6.25 In addition, the following chemicals and reagents should be on hand :

See Section 10 for the list of individual buffers and reagents formulation:
Agarose (molecular biology grade).
Orange G (re. For tracking dye in loading buffer (10.7)).
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 Tetra Acetic 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).

6.26 Dynabeads-oligo(dT)²⁵ (Cat. No 610.05, no substitution). (8.2)

6.27 Binding buffer (See section 10.3 for preparation).

6.28 Washing buffer (See section 10.4 for preparation).

6.29 Tryptose phosphate broth-glycine buffer (TPB-glycine buffer )(See section 10.1 for preparation).

6.30 TRIzol Reagent Invitrogen (Cat. No 15596-018/-026 no substitution). (8.5)

6.31 Chloroform (HPLC grade or better).

6.32 Isopropanol (HPLC grade or better).

6.33 Cold ethanol 75% in DNAse RNAse free water (stored at -20°;C).

6.34 Houde/Guévremont primers (SH-Prot-A and SH-Prot-1) for the detection of hepatitis A virus (See section 9.1 for DNA sequences).

6.35 Kageyama primers (COG2F and COG2R) for the detection of noroviruses genogroup II (See section 9.1 for DNA sequences).

6.36 Monroe primers (431,432,433 and 434) for the detection noroviruses genogroups I and II (See section 9.1 for DNA sequences).

6.37 Dynabeads Reconditioning Solution :

Sodium Hydroxide (NaOH ) (ACS grade or better).

6.38 Dynabeads Storage Buffer :

Tris[hydroxymethyl]aminomethane (e.g., Tris base molecular biology grade).
Ethylene Diamine Tetra Acetic acid (EDTA) disodium salt (molecular biology grade).
Tween20 (molecular biology grade).
Sodium Azide (ACS or better).

6.39 5 mL and 25 mL serological pipettes.

6.40 Pipet-Aid^® or equivalent.

6.41 150 mm Petri Dish.

6.42 Filtration unit (Millipore Steritop product number SCGPT05RE or equivalent).

7. Procedure

7.1 Handling of Sample Units

7.1.1 During storage and transport keep the sample units refrigerated (1°C to 5°C).

7.1.2 Analyse the sample units as soon as possible after receipt at the laboratory.

7.2 Preparation for Analysis

7.2.1 Prepare all buffers and solutions as described in section 10.

7.2.2 Prepare and verify the suitability of all controls. Include a positive and a negative controls in the RT-PCR test that is specific to the primers used (i.e. norovirus genogroup II and hepatitis A virus).

Note:
All reagents used for extraction must be at room temperature (23 °C ± 3 °C) before use (i.e. TRIzol^® reagent).

SAFETY NOTE:
ADDITIONAL PRECAUTIONS MUST BE IMPLEMENTED WHEN WORKING WITH GREEN ONIONS THAT ARE POTENTIALLY CONTAMINATED WITH NOROVIRUSES OR OTHER VIRUSES. VIRUS EXTRACTION AND CONCENTRATION STEPS FROM GREEN ONIONS ARE CARRIED OUT UNDER A BIOLOGICAL CONTAINMENT HOOD WITH A MASK, SLEEVE PROTECTORS AND DOUBLE GLOVES MADE OF NITRILE.

7.3 Virus Elution and Concentration from Green Onion Samples

Note:
Centrifuge space is a limiting factor in determining how many samples can be run at one time.

7.3.1 Cut a portion of 25 grams of green onions in small pieces (2-3 cm in length) into sterile Petri dish using a pair of sterile scissors; use mainly the green leaf part.

Note:
Virus integrity is better conserved at colder temperatures, keep the green onions in the refrigerator whenever possible.

7.3.2 Elute viruses from the surface of the cut green onions by pipetting 10 times an initial volume of 20 mL 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.

7.3.3 Transfer the residual washing solution (around 16-17 mL) from the cut green onions into an Oakridge tube. Add an equal volume of PEG 16% with 0.525 M NaCl to a chieve a final concentration of 8% (v/v). The tubes are inverted 7 times and incubate overnight in a bucket of ice (18 hours ± 1 hour) at 4 °C.

7.3.4 Centrifuge the Oakridge tube at 12,000 × g for 30 minutes at 4 °C. Discard the supernantant but do not remove the last 2-3 mL of the supernatant to avoid the loss of the pellet.

7.3.5 Add 2 mL of TRIzol^® reagent to the pellet.

SAFETY NOTE :
TRIZOL REAGENT CONTAINS PHENOL; IT SHOULD BE HANDLED CAREFULLY AND DISPOSED OF AS HAZARDOUS WASTE.

7.3.6 Vortex the Oakridge tube for 30 seconds and incubate 5 minutes at room temperature (23 °C ± 3 °C).

7.3.7 Add 0.480 mL of chloroform to the Oakridge tube and vortex for 30 seconds. Incubate 5 minutes at room temperature (23 °C ± 3 °C).

7.3.8 Transfer the whole suspension into three 2 mL micro-tubes and centrifuge at 12000 x g for 10 minutes at 4°C.

7.3.9 The upper aqueous phase is recovered and pooled back into a 15 mL centrifugation tube, adjusted with 0.5 mL volume of isopropanol, inverted by hand 5 times and incubated at room temperature for 5 minutes. Centrifuge the 15 mL tube at 5 000 X g for 5 minutes at 4°C, remove and discard the supernatant.

7.3.10 Wash the RNA pellet surface with 1 mL of cold ethanol 75% in DNAse RNAse free water (stored at -20°C) without breaking the pellet formation. Gently remove the 1 mL of cold ethanol after each wash and leave to dry for 10 to 30 minutes before adding the DNAse RNAse free water to the RNA pellet.

7.3.11 Add 400 μL of DNAse Rnase-free water to the RNA pellet to dissolve it completely. Transfer the RNA into a 1.5 mL microtube.

Note:
The total RNA concentrate can be stored at - 70°C or used immediately for purification and subsequently RT-PCR.

7.3.12 Add 400 μL of binding buffer then vortex for 30 seconds.

7.3.13 Incubate at 65° C for 2 minutes in a heatblock then cool on ice.

7.3.14 Thoroughly mix the bottle of Dynabeads-oligo(dT)₂₅ by inverting it by hand for 30 seconds.

7.3.15 Transfer 100 μL of Dynabeads-oligo(dT)₂₅ into a 2.0 mL microtube and place the tube in the magnetic bead attractor for 60 seconds with the magnet slide then discard the solution by pipetting gently being careful not to disrupt beads from the sides of the tube. Remove the magnet slide from the magnetic bead attractor.

Note:
To prevent damage to the magnetic attractor, the magnet slide must be inserted with the protruding edge of the slide downwards and with the tape side facing the tubes.

7.3.16 Add 100 μL of binding buffer to the 2.0 mL microtube and mix gently. Insert the magnetic slide into the magnetic bead attractor for 60 seconds. Discard the binding buffer using a pipette. Remove the magnetic slide.

7.3.17 Add 400 μL of binding buffer and add the 800 μL of total RNA (from step 7.3.14) to the 2.0 mL microtube containing the Dynabeads-oligo(dT)₂₅ from step 7.3.16.

7.3.18 Invert the 2.0 mL microtube twice by hand and rotate using the sample mixer at 8 rpm for 5 minutes.

Note:
If there is any solution lodged under the cap between washing steps, microcentrifuging for 2 seconds may be required prior using the magnetic bead attractor.

7.3.19 Insert the magnetic slide into the magnetic bead attractor for 60 seconds, then remove the binding buffer from the 2.0 mL microtube. Remove the magnetic slide.

Note:
If separation of magnetic beads is incomplete prolong the incubation until they separate properly.

7.3.20 Wash the magnetic beads by adding 500 μL of washing buffer to the 2.0 mL microtube and mix by inversion 5 times. Insert the magnetic slide into the magnetic bead attractor for 60 seconds, then remove and discard the washing buffer from the 2.0 mL microtube. Remove the magnetic slide.

Note:
If there is any solution lodged under the cap between washing steps, microcentrifuging for 2 seconds may be required prior using the magnetic bead attractor.

Note:
The total RNA concentrate (oyster/viruses) can be stored at - 70°C or used immediately for purification and subsequently RT-PCR.

7.3.21 Repeat washing step (7.3.19 to 7.3.20) 2 other times.

7.3.22 Remove last traces of final washing after microcentrifuging for 2 seconds and using the magnetic slide into the magnetic bead attractor.

7.3.23 Resuspend the magnetic beads with 25 μL of Rnase/DNase-free water and elute poly-A viral RNA from beads at 90°C for 2 minutes in a heatblock.

7.3.24 Separate the heated solution from beads in the magnetic bead attractor.

7.3.25 Transfer the solution (poly -A RNA) to a 0.5 mL or 1.5 mL tube.

7.3.26 Add 20 units of Rnase inhibitor to the solution.

7.3.27 Store the 0.5 mL or 1.5 mL tube at - 70 °C.

7.4 Screening RT-PCR Method for Norovirus Genogroup II or Hepatitis A Virus

7.4.1 Screen the poly-A RNA of each sample with norovirus primers and hepatitis A virus primers, described under section 9.1, using the OneStep RT-PCR kit from Qiagen.

Note:
Data from validation studies showed that the COG (Kageyama) primers offered the best analytical sensitivity. However, even if the Monroe (Anderson et al., 2001) primers are less sensitive, they offer a broader range of amplification (including Calicivirus from other Genogroup than GI and GII) and are now widely used for molecular characterization of norovirus strains (generating data for molecular epidemiology purposes) by sequencing amplicons.

7.4.2 Add 4.0 μL of the poly-A RNA (7.3.27) to 16.0 μL of RT-PCR reaction mixture (10.5).

7.4.3. Insert microfuge tubes in a thermal cycler and proceed with RT-PCR amplification according to the program described under (9.2 and 9.3).

7.4.4 Once the RT-PCR is completed, analyse the PCR product by agarose gel electrophoresis (7.5). If necessary, the amplicons can be stored at 4°C for up to one week until analysis or at -20°C for a longer time.

7.5 Agarose Gel Electrophoresis

7.5.1. Prepare a 1.5% (w/v) agarose gel in 0.5X TBE (Tris-Borate-EDTA) buffer. The agarose can be dissolved by stirring on a hot plate or by microwaving for 1 to 2 minutes using high power. Ensure that the agarose is completely dissolved (i.e. clear liquid with no particles in suspension).

7.5.2 Cool agarose around 45°C and add the ethidium bromide (EtBr) concentrated solution to have a final concentration of 0.5 μg/mL in the agarose gel (8.6). Gently mix while avoiding bubble formation.

Note:
The addition of EtBr to the gel is optional if the gel is submerged in EtBr solution after migration.

SAFETY NOTE:
EtBr IS A POTENT MUTAGEN: USE NITRILE GLOVES WHEN HANDLING.

7.5.3. Pour into a gel tray. Avoid bubble formation or bubble trapping. Add a well-forming comb and allow gel to solidify for about 20 to 30 min.

7.5.4 Prepare samples for electrophoresis: in clean microfuge tubes, mix 2 μL of Orange G tracking dye (loading buffer 10x concentrated ) with 20 μL of PCR product.

7.5.5 When the agarose gel has set, remove the comb from the tray, place the tray with gel in the electrophoresis apparatus and fill reservoir with 0.5X TBE buffer to cover gel. Gently pipet approximately 18 μL of samples (7.5.4) into the wells of the submerged gel. Pipet a sample of DNA molecular size marker (e.g., 50 bp ladder DNA) in an empty well. Include positive, negative and reagent controls.

7.5.6. Connect apparatus to power supply with cathode (-, black) situated at the top (i.e., near sample wells) and anode (+, red) at the bottom (i.e., the end) of the gel. Apply approximately 100 volts to gel and run for about 30 minutes or until the OrangeG tracking dye has spread a distance of approximately 3/4 th of the length of the gel .

Note:
The voltage and time of migration can be changed according to the power supply used.

7.5.7 Remove gel from tray and visualize DNA bands by exposure to ultraviolet light (shortwave) using a transilluminator. Gels may be photographed on Polaroid 667 film to facilitate analysis and for record keeping purposes. Alternatively a digital processing system may be used.

Note:
In the case where the EtBr has not been added directly to the gel, the gel must be remove from the tray and DNA stained by placing in ethidium bromide (EtBr) solution (0.5 μg/mL) for 30 to 45 minutes (8.6). Remove the gel from EtBr using a gel scoop, rinse briefly with tap water, and visualize DNA bands by exposure to UV light.

SAFETY NOTE:
UV LIGHT CAN CAUSE EYE DAMAGE: WEAR SAFETY GOGGLES

7.6 Reading PCR Results:

7.6.1 The amplicon (PCR product) generated by 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. Therefore, a positive PCR test will yield a DNA fragment specific to the targeted gene sequence and will appear as a band on an EtBr-stained agarose gel. The molecular size of the band can be verified by comparing its migration to that of a DNA molecular size marker (e.g., 50 bp ladder DNA) run on the same gel.

7.6.2 A negative PCR test will normally produce no visible bands in an EtBr-stained agarose gel. Although an extremely rare occurrence, any sample giving bands not corresponding to the expected amplicon size (non-specific amplification) is considered to be negative.

7.6.3 A specific band should appear for the targeted norovirus positive control and hepatitis A virus control. Absence of a positive control band invalidates the test and the samples should be re-analysed.

7.6.4 Any band corresponding to the positive control occurring in the negative or reagent control with the noroviruses or hepatitis A virus primers, indicates contamination problems with the PCR reaction mixture and the whole batch is considered suspect and should be discarded. The samples should be re-analysed using fresh reagents.

7.6.5 Any test sample showing a distinct band with the noroviruses or hepatitis A virus primers, corresponding to its corresponding positive control, is considered as a presumptive positive. The PCR product should be either purified or purified and cloned prior to sequencing to confirm the possible strain of norovirus or hepatitis A found in the sample.

*** IMPORTANT ***

Amplicons taken from the presumptive positives should be sent to a reference laboratory for sequencing, along with the food extracts (if available) for confirmation of the analysis. The reader is invited to contact the authors listed above.

7.7 Regeneration of Dynabeads-oligo(dT)₂₅ (8.2):

7.7.1 The Dynabeads-oligo(dT)₂₅ may be reused a total of four times without loss of yield. Resuspend used Dynabeads-oligo(dT)₂₅ (original volume 200 μL) in 200 μL Reconditioning Solution and transfer suspension to a new RNase-free tube.

7.7.2 Incubate at 65°C in a heatblock for 2 minutes.

7.7.3 Place tube in Dynal MPC-S attractor for at least 30 seconds and remove supernatant.

7.7.4 Wash twice in Reconditioning Solution, by repeating steps 7.7.1 and 7.7.3 twice.

7.7.5 Resuspend the Dynabeads-oligo(dT)₂₅ in 200 μL of storage buffer Oligo(dT)₂₅ .

7.7.6 Place tube on the magnet for at least 30 seconds and remove supernatant.

7.7.7 Repeat steps 7.7.5 and 7.7.6 three times.

7.7.8 Resuspend the Dynabeads-oligo(dT)₂₅ to match visually the concentration of the regenerated Dynabeads with a new lot of Dynabeads that was not regenerated with a volume of storage buffer Oligo(dT)₂₅. The Dynabeads are now reconditioned and ready for another mRNA isolation. Store in storage buffer Oligo(dT)₂₅ at 2-8°C.

Note:
Do not mix regenerated Dynabeads-oligo(dT)₂₅ with original stock suspension

8. References

8.1 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. 41:1548-1557, Vol. 41, No. 4.

8.2 Dynal Biotech insert of Dynabeads-oligo(dT)₂₅ , printed 0501, Rev. No.: 004 available at:
www.invitrogen.com/content/sfs/manuals/Dynabeads%20Oligo.pdf

8.3 Anderson, A.D., Heryford, A.G., Sarisky, J.P., Higgins, C., Monroe, S.S., Beard, R.S., Newport, C.M., Cashdollar, J.L., Fout, G.S., Robbins, D.E., Seys, S.A., Musgrave, K.J., Medus, C., Vinjé, J., Bresee, J.S., Mainzer, H.M. and Glass, R.I. 2003. A waterborne outbreak of norwalk-like virus among snowmobilers-Wyoming, 2001. Journal of Infectious Diseases. 187: 303-6.

8.4 Qiagen^® OneStep RT-PCRKit Hanbook. May 2002. For fast and efficient one-step RT-PCR. 2800 Argentia Road Unit 7 Mississauga Ontario L5N 8L2. pp:10-12. Available at: www1.qiagen.com/literature/handbooks/PDF/PCRAndReverseTranscription/
KitsAndEnzymes/RTPCR_OneStep/1020892HBRTPCR_05202.pdf

8.5 Invitrogen^® TRIzol. Product 15596-026. (2 pages) available at:
http://www.invitrogen.com/content/sfs/manuals/15596026.pdf

8.6 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, USA, p 6.15.

8.7 Guévremont, E, Brassard, J., Houde, A., Simard, C., 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. Volume 134, Issues 1-2, June 2006, Pages 130-135.

9. PCR Primers and Temperature Cycling Program

9.1 PCR Primers

Screening PCR: Oligonucleotide primers for the PCR are based on the most conserved region in the norovirus genome.

Kageyama primers (8.1) for the detection of genogroup II norovirus (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 (8.3) for the detection of genogroups I and II noroviruses (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 (8.7) for the detection of HAV (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 MixBase Definitions
Letter	Nucleotides	Letter	Nucleotides
R	A, G	H	A, C, T
Y	C, T	B	C, G, T
M	A, C	V	A, C, G
K	G, T	D	A, G, T
S	C, G	N	A, C, G, T
W	A, T	X	A, C, G, T

NOTE 1:
Synthesis of oligonucleotide primers can usually be contracted out to a local university or, alternatively, many biotechnology firms offer a custom synthesis service. If assistance is required in this matter, contact the authors.

NOTE 2:
For better results, it is recommended to have primers that have been purified by High Pressure/Performance Liquid Chromatography (HPLC).

9.2 Temperature cycling program for all Norovirus primers specific for genogroups II

The thermal cycler program for Monroe and Kageyama primers should be set to the following sequence of cycling parameters :

Step No	Process	Time	Temperature	Remarks
1	Reverse transcription	30 minutes	50°C
2	Initial PCR activation step	15 minutes	95°C	(HotStarTaq DNA Polymerase) Omniscript and Sensiscript Reverse Transcriptases are inactivated and the cDNA template is denatured
3	40 cycles of (3.1+3.2+3.3)
	3.1 Melting	45 seconds	94°C
	3.2 Annealing	30 seconds	52°C
	3.3 Extension	45 seconds	72°C
4	Final elongation	10 minutes	72°C

Note:
The use of thermal cyclers other than the models stated above may alter the performances of the PCR, and it may be necessary, for the user, to optimize cycling parameters for different models.

9.3 Temperature Cycling Program for Primers Specific for Hepatits A Virus

The thermal cycler program should be set to the following sequence of cycling parameters:

Step No	Process	Time	Temperature	Remarks
1	Reverse transcription	30 minutes	50°C
2	Initial PCR activation step	15 minutes	95°C	(HotStarTaq DNA Polymerase) Omniscript and Sensiscript Reverse Transcriptases are inactivated and the cDNA template is denatured
3	40 cycles of (3.1+3.2+3.3)
	3.1 Melting 3.2 Annealing 3.3 Extension	45 seconds 30 seconds 45 seconds	94°C 52°C 72°C
4	Final elongation	10 minutes	72°C

10. REAGENTS

10.1 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 a volume of 800 mL, dissolve, adjust the pH to 9.5 and complete to 1.0 L . The solution is then filter sterilized using a 0.22 μM filter unit.

Note 1:
The final pH must be at 9.5 ± 0.1 after filter sterilisation.

NOTE 2:
The TPB-glycine buffer pH 9.5 is stable for 9 months at 4°C.

10.2 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 a volume of 500 mL, dissolve and autoclave, 121°C 15 minutes.

Note:
The PEG 16 % is stable for 9 months at 4°C.

10.3 Binding

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).

Example of preparation of 50 mL of binding buffer from 0.1 M Tris-HCl, 8 M LiCl, 0.5 M EDTA and sterile distilled water (DNAse RNAse Free) from readily commercial solutions:
0.020 M of Tris-HCl, pH 7.5 * 50 mL = 0.1 M Tris-HCl * x mL
x mL = 10 mL of 0.1 M Tris-HCl

1.0 M of LiCl * 50 mL = 8 M LiCl * x mL
x mL = 6.25 mL of 8.0 M LiCL

0.002 M of EDTA * 50 mL = 0.5 M EDTA * x mL
x mL = 0.200 mL of 0.5 M EDTA

Complete with sterile distilled water (DNAse RNAse Free) to have 50 mL.
x mL = 33.55 mL of water

In a sterile 50 mL centrifugation tube, mix all reagents and aliquot in smaller volumes for storing

Note :
The binding buffer is stable for 12 months at room temperature (23°C ± 3°C).

10.4 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).

Example of preparation of 50 mL of washing buffer from 0.1 M Tris-HCl, 8 M LiCl, 0.5 M EDTA and sterile distilled water (DNAse RNAse Free) from readily commercial solutions:

0.010 M of Tris-HCl, pH 7.5 * 50 mL = 0.1 M Tris-HCl * x mL

x mL = 5 mL of 0.1 M Tris-HCl

0.15 M of LiCl * 50 mL = 8 M LiCl * x mL

x mL = 0.938 mL of 1.0 M LiCL

0.001 M of EDTA * 50 mL = 0.5 M EDTA * x mL

x mL = 0.100 mL of 0.5 M EDTA

Complete with sterile distilled water (DNAse RNAse Free) to have 50 mL.
x mL = 43.962 mL of water

In a sterile 50 mL centrifugation tube, mix all reagents and aliquot in smaller volumes for storing.

Note :
The binding buffer is stable for 12 months at room temperature (23 °C ± 3°C).

10.5 Qiagen OneStep RT-PCR Kit

All stock solutions are also stored at -20°C until use. The following is a recipe for preparing a large batch equivalent to 20 reactions.

Note :
All reagents, Dnase RNase-free water, pipet tips and other materials coming into contact with samples or RT-PCR reagents should be sterile or autoclaved prior to use to remove any DNAses and/or other contaminants. To avoid contamination problems, all reagents should be prepared in a laminar flow cabinet which has never been exposed to noroviruses or norovirus PCR products and hepatitis A virus or PCR products. To avoid any non-specific amplifications, the mix should be prepared by putting all the reagents on ice or on a cooled rack.

RT-PCR Components	Initial concentration	Stock solutions required for 20 reactions tubes using Monroe primers	Volume per tube	Final Concentration
RNase-free water	---------	112.0 μL	5.6 μL	--------
5x QIAGEN OneStep RT-PCR Buffer	5x	80.0 μL	4 μL	1 x
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 Volume		320 μL	16 μL	--------
Distribute per tube		16 μL	--------	--------
Template per tube		4 μL	--------	--------

 

RT-PCR Components	Initial concentration	Stock solutions required for 20 reactions tubes using Kageyama primers for genogroups II	Volume per tube	Final Concentration
RNase-free water	---------	160.0 μL	8 μL	--------
5x QIAGEN OneStep RT-PCR Buffer	5x	80.0 μL	4 μL	1 x
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 Volume		320 μL	16 μL	--------
Distribute per tube		16 μL	--------	--------
Template per tube		4 μL	--------	--------

 

RT-PCR Components	Initial concentration	Stock solutions required for 20 reactions tubes using Houde/Guévremont primers for HAV	Volume per tube	Final Concentration
RNase-free water	---------	160.0 μL	8 μL	--------
5x QIAGEN OneStep RT-PCR Buffer	5x	80.0 μL	4 μL	1 x
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 Volume		320 μL	16 μL	--------
Distribute per tube		16 μL	--------	--------
Template per tube		4 μL	--------	--------

10.6 5 X Tris-Borate-EDTA - (TBE) buffer or commercially available

Tris Base (molecular biology grade). 54.0 g
Boric Acid (molecular biology grade). 27.5 g
EDTA disodium (molecular biology grade). 3.75 g
Add distilled water to a volume of 800 mL, dissolve, complete to 1.0 L. This buffer is used at a 1:10 dilution (TBE 0.5 X Buffer) in distilled water.
The pH of the 0.5 X buffer should be 8.3. Do not adjust the pH.

10.7 Tracking dye / Loading Buffer or commercially available

10X Orange G, sodium salt 0.025 g 0.25 % (w/v)
Glycerol 4 mL 40% (w/v)

Sterile distilled water (DNAse RNAse Free) 6 mL
Mix all the ingredients thoroughly, sterilized by autoclaving at 121°C for 15 minutes or filter sterilized using a 0.22 μM filter unit and store in 1.0 mL aliquot at -20°C.

Note :
The tracking dye / loading buffer binding buffer is stable for 12 months at - 20°C.

10.8 DNA molecular size marker (commercially available)

Although many types of DNA size marker preparations are available from different suppliers, the 50 bp ladder DNA marker provides a useful range of DNA fragment sizes and facilitates the "sizing" of PCR amplicons generated in this reaction.

10.9 Reconditioning solution for Dynabeads-oligo(dT)₂₅

0.1 M NaOH (ACS grade or better).
Example of preparation of 50 mL of stock solution of 1 M NaOH:
Weight 2.0 g of NaOH and complete with sterile distilled water (DNAse RNAse Free) to have 50 mL using a 50 mL sterile centrifugation tube. The solution is then filter sterilized using a 0.22 μM filter unit.
Make a 1:10 dilution using distilled water (DNAse RNAse Free) to have a 0.1M NaOH working solution.

Note :
This solution is stable for 6 months at room temperature (23°C ± 3°C).

10.10 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% Tween20 (molecular biology grade commercially available).
0.02% Sodium Azide (NaN3) (ACS grade)

Example of preparation of 50 mL of storage buffer from 1.0 M Tris-HCl, 0.5 M EDTA, tween solution, Sodium Azide powder and sterile distilled water (DNAse RNAse Free) from readily commercial solutions:
0.250 M of Tris-HCl, pH 7.5 * 50 mL = 1.0 M Tris-HCl * x mL
x mL = 12.5 mL of 0.1 M Tris-HCl

0.020 M of EDTA * 50 mL = 0.5 M EDTA * x mL
x mL = 2.0 mL of 0.5 M EDTA
Add 50 μL of tween 20.
Add 0.01 g of sodium azide (NaN3)
Add 33.55 mL of sterile distilled water (DNAse RNAse Free).
In a sterile 50 mL centrifugation tube, mix all reagents and aliquot in smaller volumes for storing.

Note :
The storage buffer is stable for 6 months at room temperature (23°C ± 3°C).