Concentration of norovirus genogroups I and II from contaminated oysters and their detection using the reverse-transcriptase polymerase chain reaction

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OPFLP-01

March 2010

Laboratory Procedure

Health products and food branch

Ottawa

Yvon-Louis Trottier¹, Alain Houde², Enrico Buenaventura³, Peter Müller¹,
Julie Brassard², Katie Christensen³, and Jennifer Liu³

¹ St-Hyacinthe Laboratory,
Canadian Food Inspection Agency,
3400 Casavant Blvd W ,
Saint-Hyacinthe, QC ,Canada
J2S 8E3

² Agriculture and Agri-Food Canada,
Food Research and Development Centre,
3600 Casavant Blvd.West,
St. Hyacinthe, QC, Canada, J2S 8E3

³ Burnaby Laboratory,
Canadian Food Inspection Agency,
W2250 Boundary Road, Burnaby, BC, Canada
V5M 4L9

Microbiological Methods Committee
Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Postal Locator: 2204E
HPFB, Ottawa, Ontario, K1A 0K9

E-mail : Micro_methods_committee@hc-sc.gc.ca

1. Application

This method is applicable to the concentration and detection of Norovirus belonging to genogroups I and II from contaminated oysters. It has been shown to give acceptable results in bivalve molluscan shellfish such as oysters and mussels. This method will not differentiate between infectious and non-infectious viruses.

2. Description

Initially, this method has shown to produce satisfactory results with artificially and naturally contaminated (spiked) oysters and mussels with genogroup I and II with Noroviruses. This method has also been used by the USFDA in a round robin study. The initial procedure was modified and improved for the routine detection of Norovirus genogroups I and II.

3. Principle

Following a slightly modified procedure, described by Kingsley and Richards (8.1), viral particles are concentrated by homogenization of the digestive glands by sonication in glycine buffer, followed by a polyethylene glycol (PEG) precipitation step. Total RNA is then extracted with TriReagent ®. The bulk of the total RNA extracted is oyster. Enrichment of viral RNA from total RNA is performed using oligo-dT coated magnetic beads. These magnetic beads bind the polyadenylated (poly-A tail) messenger RNA (mRNA) of Noroviruses. The final extract is then subjected to a reverse-transcriptase polymerase chain reaction (RT-PCR) procedure which amplifies a specific fragment depending on the primers used (i.e. Norovirus genogroups I / II, and the actin gene target for oysters).

4. Definition of Terms

See Appendix A of Volume 3.

5. Collection of Samples

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 17025:1999 (or latest version). General requirements for the competence of testing and calibration laboratories".

1) Thermal cycler (Eppendorf ^® Mastercyler ^® gradient or equivalent).

2) Microwave oven or hot plate.

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

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

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

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

7) Standard heatblocks (VWR scientific products or equivalent) capable of accommodating 1.5 mL microfuge tubes and capable of maintaining a temperature of 37°C, 65°C, 70°C, 90°C and 95°C.

8) Waterbath capable of maintaining a temperature of 37°C.

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.

9) Vortex mixer.

10) Microfuge tubes - 2.0 and 1.5 mL capacity.

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

12) 50 mL (Polypropylene Copolymer Oak Ridge centrifugation tubes or Polypropylene centrifugation tubes).

13) IEC DPR-6000 refrigerated centrifuge or equivalent.

14) IEC DPR-6000 rotors for 50 mL and 15 mL tubes or equivalent.

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

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

17) Dynal ^® Biotech Sample Mixer (product number 947.01 or equivalent).

18) Sonicator with probe (Sonics & Materials 375 Watt Model or equivalent).

19) Microfuge.

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

21) Timer.

22) Container for Ice.

23) Magnetic stirrers.

24) Freezers capable of maintaining - 20 °C and - 70°C.

25) Qiagen OneStep RT-PCR Kit (product numbers 210210 or 210212, no substitution). (8.6)

26) 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).
Ethidium bromide (EtBr) (molecular biology grade).
Rnase inhibitor.
Boric acid (molecular biology grade).
Water DNAse RNAse free (molecular biology grade).
Polyethylene Glycol (PEG) 8000 (molecular biology grade).
Sodium Chloride (NaCl) (molecular biology grade).

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

28) Binding buffer (See section 10.3 for preparation).

29) Wash buffer (See section 10.4 for preparation).

30) Glycine buffer (See section 10.1 for preparation).

31) TriReagent Sigma (Cat. No T9424, no substitution). (8.7)

32) Chloroform (HPLC grade or better).

33) Isopropanol (HPLC grade or better).

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

35) Kageyama primers (COG1F and COG1R) for the detection of genogroup I Noroviruses (See section 9.1 for DNA sequence).

36) Kageyama primers (COG2F and COG2R) for the detection of genogroup II Noroviruses(See section 9.1 for DNA sequence).

37) Monroe primers (431,432,433 and 434) for the detection of genogroups I and II Noroviruses(See section 9.1 for DNA sequence).

38) Actin-A and Actin-R primers for the detection of oyster actin mRNA (See section 9.1 for DNA sequence).

39) Dynabeads Reconditioning Solution :
Sodium Hydroxide (NaOH ) (ACS grade or better).

40) 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).

7. Procedure

7.1 Handling of Sample Units

7.1.1 During storage and transport keep the sample units refrigerated (0 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 at the RT-PCR test that is specific to the primers used (i.e. Noroviruses genogroups I or II ).

7.3 Virus extraction and concentration from oyster samples

Safety note :
Additional precautions must be implemented when working with oysters/mussels that are potentially contaminated with noroviruses. virus extraction and concentration steps from oysters are carried out under a biological containment hood with a mask, sleeve protectors and double gloves made of nitrile.

7.3.1 Remove one shell of each oyster, drain the liquor (mantle fluid) into a discard container, and place part of the digestive gland (minus the adductor muscle that is left attached to the shell) into a tared 50 mL sterile centrifugation tube. Sample size should be ideally 1.5 grams ± 0.1 gram of digestive gland.

Note:
Higher viral numbers are usually found in the stomach and digestive divercula as a consequence of bioaccumulation by the oyster. In order to help in the location and identification of these biological features see Figures 1, 2 3, and 4 at the end of the proceure.

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

7.3.2 Add 12 mL of chilled sterile glycine buffer to the 50 mL centrifugation tube and place the tube in a small beaker filled with ice and place the probe into the tube.

Safety note :
ULTRASOUNDS CAN CAUSE PERMANENT EAR DAMAGE: WEAR EAR PROTECTION (E.G. EAR PLUGS AND/OR EAR MUFFS).

7.3.3 Homogenize for 1 minute the sample while rotating gently the probe inside the 50 mL centrifugation tube. Adjust the Sonic sonicator settings to 40 percent Dutty and 3.5 output control (chose equivalent settings for other sonicators).

Note:
Virus integrity is better conserved at colder temperatures, keep samples in ice whenever possible.

Note:
Always clean the probe with 70 % alcohol between samples and wipe dry.

7.3.4 Mix the 50 mL centrifugation tube by inversion and incubate at 37°C for 30 minutes in a waterbath.

7.3.5 Centrifuge the homogenate at 3700 X g for 120 minutes at 4°C, decant the supernatant in a sterile 50 mL centrifugation tube and add an equal volume of PEG 16%. Invert the tube 7 times and incubate overnight in bucket of ice (18 hours ± 1 hour).

7.3.6 Centrifuge the 50 mL centrifugation tube at 3700 X g for 14 minutes at 4°C and discard the supernatant.

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

7.3.7 Resuspend the pellet with 5 mL of TriReagent and transfer it into a 15 mL centrifugation tube.

Safety note :
Trireagent contains phenol it should be handled carefully and disposed of as hazardous waste.

7.3.8 Vortex the 15 mL tube for 30 seconds and incubate 5 minutes at room temperature (23°C ± 3°C).

7.3.9 Add 1.2 mL of chloroform to the 15 mL centrifugation tube and vortex for 30 seconds. Incubate 5 minutes at room temperature (23°C ± 3°C).

7.3.10 Centrifuge the 15 mL centrifugation tube at 5000 X g for 24 minutes at 4°C. Transfer the upper aqueous layer to a clean 15 mL centrifugation tube and add 0.5 volume of isopropanol and invert 5 times to mix. Incubate for 5 minutes at room temperature.

Note:
See Figure 5 to visualize an example of the three different layers obtained after the centrifugation procedure.

7.3.11 Centrifuge the 15 mL tube at 5 000 X g for 5 minutes at 4°C , remove and discard the supernatant.

7.3.12 Wash the RNA pellet surface with 5 mL of cold ethanol 75% in DNAse RNAse free water (stored at -20°C) without breaking the pellet formation and leave to dry for 5 to 10 minutes before adding the DNAse RNAse free water to the RNA pellet.

7.3.13 Add 500 µL of DNAse Rnase-free water to the RNA pellet to dissolve it completely. Transfer the RNA in a 1.5 mL microtube.

Note:
The sample can be heated at 60°C in a heat block and vortex to dissolve if necessary.

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

7.3.14 Transfer 400 µL of the total RNA to a 1.5 mL microtube and add 400 µL of Binding buffer then vortex for 30 seconds.

7.3.15 Incubate at 65° C for 2 minutes in a heat block then cool on ice.

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

7.3.17 Transfer 100 µL of Dynabeads-oligo(dT)₂₅ to 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.

7.3.18 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.

7.3.19 Add 400 µL of binding buffer and add the 800 : L of total RNA (from step 7.3.14) to the 2.0 mL microtube containing the 100 µL of Dynabeads-oligo(dT) 25.

7.3.20 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 wash steps, microcentrifuging for 2 seconds may be required prior to using the magnetic bead attractor.

7.3.21 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.

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

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

7.3.23 Repeat wash step (7.3.22) 2 other times.

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

7.3.25 Resuspend the magnetic beads with 25 µL of Rnase/DNase-free water and elute PolyA viral RNA from beads at 90°C for 2 minutes in a heat block.

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

7.3.27 Transfer the solution (Poly A RNA ) to a 0.5 mL or 1.5 mL tube.

7.3.28 Add 20 units of Rnase inhibitor to the solution.

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

7.4 Screening RT-PCR Method for Noroviruses (i.e. detection of oyster actin mRNA, Norovirus genogroups I and II)

7.4.1 Screen the poly-A RNA of each sample with the 3 sets of Norovirus primers and the actin house keeping gene for oysters (9.1) using the OneStep RT-PCR kit from Qiagen.

7.4.2 Add 5.0 µL of the poly A RNA (7.3.29) to 20.0 µL of 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.

7.4.4 After 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.

7.5 Agarose gel electrophoresis

7.5.1. Prepare a 3.0% (w/v) agarose gel in 0.5 X 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 concentrated solution to have a final concentration of 0.5 µg/mL in the agarose gel (8.8). 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 :
Ethidium bromide (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 set for about 20 to 30 min.

7.5.4. Prepare samples for electrophoresis: in clean microfuge tubes, mix 2.5 µL of tracking dye
(loading buffer 10x concentrated ) with 25 µL of PCR product.

7.5.5. Whenthe agarose gel has set,remove the comb from the tray, place the tray with gel in the electrophoresis apparatus and fill reservoir with 0.5 X 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 135 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 (8.8). 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 the Actin-A/R primers, COG1F/R primers, COG2F/R and 431,432,433,434 primers are double stranded DNA fragments of 257 bp, 85 bp, 98 bp and 213 bp respectively. Therefore, a positive PCR test will yield a DNA fragment specific to the targeted gene sequence and will appear as an intense 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. ( See Figure 6 - Guide for the reading of RT-PCR reactions.)

7.6.2 A negative PCR test will normally not produce any 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. Absence of a positive control band invalidates the test and the samples should be re-analyzed. In cases where a positive reaction is observed for norovirus but no reaction is observed for the actin gene may be indicative of a cross contamination between samples.

7.6.4 Any band corresponding to the positive control occurring in the negative or reagent control with the Norovirus 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 a fresh reagents.

7.6.5 Any test sample showing a distinct band with the Norovirus primers, corresponding to its 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 found in the oyster.

Note:
All air bubbles MUST be removed before proceeding to the next step. If air bubbles remain, repeat vigorous striking of the wells onto an absorbent paper towel on a flat surface until eliminated.

7.7 Regeneration of Dynabeads-oligo(dT)₂₅ (8.4):

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 drybath for 2 minutes.

7.7.3 Place tube in Dynal ^® MPC-S 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 : L 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 7.5.6 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 Kingsley, D.H. and Richards, G.P. 2001. Rapid and efficient extraction method for reverse transcription-PCR detection of hepatitis A and Noroviruses in shellfish. Applied and Environmental Microbiology 67:4152-4157.

8.2 Shieh, Y.C. Baric, R.S., Woods, J.W., and Calci, K.R. 2003, Molecular surveillance of enterovirus and norwalk-like virus in oysters relocated to a municipal-sewage-impacted gulf estuary. Applied and Environmental Microbiology. 69:7130-7136

8.3 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 Noroviruses based on real-time quantitative reverse transcription-PCR. Journal of Clinical Microbiology. 41:1548-1557, Vol. 41, No. 4

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

8.5 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.

8.6 Qiagen^®. 2002. Qiagen^® OneStep RT-PCRKit Hanbook. 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

8.7 Sigma^®. 1999. Technical Bulletin MB-205. Product Information TRI-Reagent Product T 9424. (6 pages) available at www.sigmaaldrich.com/sigma/bulletin/t9424bul.pdf

8.8 Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989, Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, p 6.15

8.9 Howard, D.W. and Smith, C.S. 1983, Histological techniques for marine bivalve mollusks. NOAA Techncial Memorandum NMFS-F/NEC 25:97.

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 (8.3) for the detection of genogroup I Norovirus (85 bp fragment):

COG1F - forward - 20 bases = 5'-CGY TGG ATG CGN TTY CAT GA- 3'

COG1R - reverse - 22 bases = 5'-CTT AGA CGC CAT CAT CAT TYA C-3'

Kageyama primers (8.3) 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 (8.5) for the detection of genogroups I and II Norovirus (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'

Actin house keeping gene target for oysters (8.2):

Primers for the detection of oyster actin mRNA (257 bp fragment):

Actin-A - forward - 23 bases = 5'-TGG AAT CTG CYG GWA TCC ATG AA-3'

Actin-R - reverse - 23 bases = 5'-CCG ATC CAG ACG GAG TAT TTC CT-3'

colspan="4"> 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:
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.

9.2 Temperature cycling program for all Norovirus primers specific for genogroups I and II andfor theactin house keeping gene target for oysters

The thermal cycler program should be set for 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 (a + b + c)	a. Melting	30 seconds	94°C
	b. Annealing	30 seconds	52°C
	c. Extension	45 seconds	72°C
4	Final elongation	5 minutes	72°C

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

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

10. Reagents

10.1 Glycine buffer pH 9.5

7.50 g Glycine (molecular biology grade).

17.55 g NaCl (molecular biology grade).

Add distilled water to a volume of 800 mL , dissolve and adjust the pH to pH 9.

Complete to 1.0 L and autoclave, 121°C 15 minutes or adjust at pH 9.5 and filter sterilize.

Note:
The final pH must be at 9.5 ± 0.1 after autoclaving.

Note:
The glycine buffer pH 9.5 is stable for 9 months at 4 °C.

10.2 16 % polyethylene glycol (PEG)

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 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).

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 Wash 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 wash 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 wash 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. To avoid any non-specific amplifications, the mix should be prepared by putting all the reagents on ice or on a refrigerated rack.

>RT-PCR Components	>Initial concentration	Stock solutions required for 20 reactions tubes using Monroe primers	Volume per tube	Final Concentration
RNase-free water		140.0 µL	7 µL
5x QIAGEN OneStep RT-PCR Buffer	5x	100.0 µL	5 µL	1 x
dNTP Mix	10 mM/of each dNTP	20.0 µL	1 µL	400 µM
Primer 431 - forward	10 µM/per primer	30.0 µL	1.5 µL	0.6 µM
Primer 432 - forward	10 µM/per primer	30.0 µL	1.5 µL	0.6 µM
Primer 433 - reverse	10 µM/per primer	30.0 µL	1.5 µL	0.6 µM
Primer 434 - reverse	10 µM/per primer	30.0 µL	1.5 µL	0.6 µM
QIAGEN OneStep RT-PCR Enzyme Mix		20.0 µL	1 µL
Total Volume		400 µL	20 µL
Distribute per tube		20 µL
Template		5 µL

 

>RT-PCR Components	>Initial concentration	>Stock solutions required for 20 reactions tubes using Kageyama primers for genogroups I or II and actin primers	>Volume per tube	>Final Concentration
RNase-free water		180.0 µL	9 µL
5x QIAGEN OneStep RT-PCR Buffer	5x	100.0 µL	5 µL	1 x
dNTP Mix	10 mM/of each dNTP	20.0 µL	1 µL	400 µM
Primers COG1F -forward or COG2F - forward	10 µM/per primer	30.0 µL	1.5 µL	0.6 µM
Primers COG1R - reverse or COG2R - reverse	10 µM/per primer	30.0 µL	1.5 µL	0.6 µM
Primer Actin-A - forward	10 µM/per primer	10.0 µL	0.5 µL	0.2 µM
Primer Actin-R - reverse	10 µM/per primer	10.0 µL	0.5 µL	0.2 µM
QIAGEN OneStep RT-PCR Enzyme Mix		20.0 µL	1 µL
Total Volume		400 µL	20 µL
Distribute per tube		20 µL
Template		5 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, sterilize by autoclaving at 121°C for 15 minutes or filter sterilize 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 centrifugation tube. The solution is then filter sterilize.

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 ( NaN 3 ) (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 (NaN 3)

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).