Government of Canada
Gouvernement du Canada
Lab orato ry Proc edu re
OPFLP -01 March 2010
HEALTH PRODUCTS AND FOOD BRANCH O TT AW A CONC ENTRATION OF NO ROVIRUS GENO GROU PS I AND II FROM CONT AMINATED OYSTERS AND THEIR DETECTION USING THE REVERSE-TRANSCRIPTASE POLYMERASE CHAIN REACTION Yv on-Louis T rottier 1, Alain Houde 2, Enrico Buenaventura 3, Peter M üller 1, Julie Brassard 2, Katie Christensen 3, and Jennifer Liu 3 1
St-H yacin the L abo rato ry ,Canadian Food Inspection Agency , 3400 Casavant Blvd W , Saint-Hyacinthe, QC ,Canada J2S 8E3
2
A g ri cu lt ur e a n d A g ri -F o od C a na d a, Fo o d R e s ea rc h an d D ev el op m e n t C e n tr e, 3 6 00 C a sa va n t B lv d. W e st , S t. H y ac in th e , Q C , C a na d a, J 2 S 8 E 3
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 :
[email protected]
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 noninfec tious 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 de tection of N orov irus geno grou ps I and II.
3.
PRINCIPLE Following a slightly mod ified proce dure, described by Kingsley and R ichards (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 (m RNA) of N oroviruses. T he fina l extract is then subjec ted to a reverse-tra nscripta se polym erase chain reaction (RT-PCR ) procedure which amplifies a specific fragment depending on the primers used (i.e.
Publishe d on the F ood Directora te’s (H ealth C ana da's) website a t http://www.hc-s c.gc .ca/fn -an/res-rech/an aly-m eth/m icrobio/index_e .htm l
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OPFLP-01 March 2010
Norovirus gen ogroups I / II, and the actin gene target for oysters). 4.
DEFINITION OF TERMS See Appendix A of Volume 3.
5.
COLLECT ION OF SAMPLES
or
The num ber of samples will have to be determined on the basis of the client needs (e.g. Data collection surveys) or investigational purpose s (e.g. Outbreak s).
6.
MATERIALS AND SPECIAL EQUIPMENT Note: The Laboratory Supervisor m ust ensure that co m pletion of the analysis, described in this method, must be don e in ac cordan ce w ith the Inte rnational Standard referred to as "ISO/IEC 17025:1999 (or latest vers ion). G ene ral requirem ents for the com pete nce of tes ting an d ca libration labora tories".
Note:
1)
The rm al cycler (Eppen dorf ® 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 doc um entation system (optional, for photographic record s), including polaroid cam era (han d-he ld or fixe d), ho od a nd P olaroid™ 667 film o r equ ivalent.
6)
Adjustable micropipettors: to cover range of volumes: 0.5 to 10 :L, 10 to 100 :L, and 10 0 to 1000 :L with specific filtered pipet tips.
7)
Standard heatblocks (VW R scientific products or equivalent) capable of accomm odating 1.5 mL microfuge tubes and capable of maintaining a temperature of 37/C, 65/C, 70/C, 90/C and 95/C.
8)
W aterbath capable of maintaining a temperature of 37/C.
It is the responsibility of each laboratory to ensure that the block heaters or water baths are maintained at the recomm ended temperatures. W here 37/C is recomm ended the wate rbath may be at 37/C +/- 1/C. For all other tempera tures it may be +/- 2/C. 9)
Vortex m ixer.
10)
Microfuge tubes - 2.0 and 1.5 m L capacity.
11)
SORV ALL ® RC-5B Plus™ refrigerated centrifuge with rotors number SS-34 (8 X 50 mL Polypropylen e Copo lyme r Oak Ridg e tubes) or eq uivalen t.
12)
50 mL (Polypropylene Copolymer Oak Ridge centrifugation tubes or Polypropylene centrifugation tubes).
13)
IEC DP R-6 000 refrige rated cen trifuge or eq uivalen t.
14)
IEC DP R-6 000 rotors for 50 m L an d 15 m L tubes o r equ ivalent.
15)
15 mL polypropylene centrifugation tubes. (Sarstead ® product num ber 62.554.002 or better).
16)
Dynal® Mag netic bead attractors m odel MPC -S (product num ber 120.20, no su bstitution).
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OPFLP-01 March 2010
17)
Dynal® Biotech Sam ple Mixer (produc t num ber 947.01 or eq uivalent).
18)
Sonicator with probe (Son ics & Ma terials 375 W att Model or equivalent).
19)
Microfuge.
20)
Tub es for PC R - thin wall 0.2 mL or 0.5 m L capac ity (depending on therm al cycler model).
21)
Tim er.
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 ch em icals and re age nts s hou ld be o n ha nd. See Section 10 for the list of individual buffers and reagents formulation: Agaros e (m olecular biology grade). Orang e G (re. For trac king dye in loading buffer (10.7)). DN A Ladd er 50 bp (or equ ivalent). Ethidium brom ide (E tBr) (m olecular biology gra de). Rnas e inhibitor. Boric acid (m olecular biology grade). W ater DNA se RN Ase free (m olecular biology grade). Polyethylene G lycol (PEG ) 8000 (m olecular biology grade). Sodium Chloride (NaC l) (molecular biology grade).
27)
Dynabeads-oligo(dT) 25 (Cat. No 610 .05, no substitution).
28)
Binding buffer (See se ction 10.3 for preparation).
29)
W ash buffer (Se e section 10.4 for prepara tion).
30)
Glycine buffer (See sec tion 10.1 for preparation).
31)
TriReagent Sigma (Cat. No T9424, no substitution). (8.7)
32)
Chloroform (HPLC grade or better).
33)
Isopropano l (HPLC g rade or better).
34)
Cold ethanol 75% in DNAse RNA se free water (stored at -20/C).
35)
Kageyama primers (COG 1F and COG 1R) for the detection of genogroup I Noroviruses (See section 9.1 for DN A sequ ence).
36)
Kageyama primers (COG 2F and COG 2R) for the detection of genogroup II Noroviruses(See section 9.1 for DN A sequ ence).
37)
Mo nroe prim ers (431 ,432 ,433 and 434 ) for the de tection of ge nog roup s I and II Noroviruses (See sec tion 9.1 for DNA se quenc e).
38)
Actin-A and Actin-R primers for the detection of oyster actin mRNA (See section 9.1 for DNA seque nce).
39)
Dynabeads Reconditioning Solution :
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OPFLP-01 March 2010
Sodium Hydroxide (NaO H ) (AC S grade o r better). 40)
7.
Dynabea ds Storage Buffer : Tris[hydroxyme thyl]am inom ethane (e.g., Tris base m olecular biology grade). Ethylene Diamine Tetra Acetic acid (EDTA) disodium salt (molecular biology grade) Twe en20 (m olecular biology grade). Sodium Azide (ACS o r better).
PROCEDURE 7.1
7.2
7.3
Han dling of S am ple Un its 7.1.1
During storage and transport keep the sample units refrigerated (0 to 5/C).
7.1.2
An alyse the sam ple units as soon as possible after receipt at the laborato ry.
Preparation fo r Analysis 7.2.1
Prepare all buffers and solutions as described in section 10.
7.2.2
Prepare a nd verify the suitability of all controls. Include a pos itive and a ne gative controls at the RT-PCR test that is specific to the primers used (i.e. Noroviruses gen ogro ups I or II ).
Virus extraction and concentration from oyster samples
SAFET Y N OT E:
7.3.1
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. Rem ove one s hell of each oyster, drain the liquor (man tle fluid) into a discard co ntainer, and place part of the digestive glan d (m inus the addu ctor m usc le that is left attached to the shell) in to a tared 50 m L sterile centrifu gation tube. S am ple size should be ideally 1.5 grams ± 0.1 gram of digestive gland.
Note :
Higher viral num bers are usually found in the stoma ch 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 :
Ce ntrifug e sp ace is a limiting fac tor in de term ining how m any sam ples can be ru n at one tim e. 7.3.2
SAFETY NOTE:
7.3.3
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. UL TR ASO UN DS CAN CAU SE P ER M ANE NT EAR D AM AGE : W EAR EAR PRO TEC TIO N (e.g. EAR P LUG S AND /OR E AR M UFF S). Hom ogenize for 1 minute the sample while rotating gently the probe inside the 50 mL cen trifuga tion tub e. Ad just the So nic sonicator s ettings to 40 perc ent D utty and 3.5 outp ut control (chos e eq uivalen t settings for other son icators).
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OPFLP-01 March 2010
Note :
Virus integrity is better conserved at colder temperatures, keep samples in ice whenever possible.
Note :
Alw ays clean the pro be with 70 % alcohol betw een sam ples and wipe dry.
Note:
7.3.4
Mix the 50 mL centrifugation tube by inversion and incubate at 37 /C for 30 minutes in a wate rbath .
7.3.5
Ce ntrifug e the hom oge nate at 3700 X g for 1 20 m inutes 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
Ce ntrifug e the 50 m L ce ntrifug ation tube a t 3700 X g for 1 4 m inutes at 4/C and discard the supe rnata nt.
All reagents used for extraction must be at room temp erature (23 /C ± 3 /C) before use (i.e. TriReag ent). 7.3.7
SAFEY NO TE:
Resuspend the pellet with 5 mL of TriReagent and transfer it into a 15 mL centrifugation tube. TRIREAGENT CONTAINS PHENOL IT SHOULD BE HANDLED CAREFULLY AND DISPOSED OF AS HAZARDOU S 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 Ce ntrifug e the 15 m L ce ntrifug ation tube a t 5000 X g for 2 4 m inutes 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 m ix. Incubate for 5 minutes at room temperature. Note :
See Figure 5 to visualize an example of the three different layers obtained after the centrifugation proc edu re. 7.3.11 Ce ntrifug e the 15 m L tube at 5 000 X g for 5 m inutes at 4/C , remove and discard the sup erna tant. 7.3.12 W ash the R NA pellet su rface with 5 m L of cold ethanol 75% in D NAs e R NAs e free water (stored at -20/C) without breaking the pellet formation and leave to dry for 5 to 10 m inutes befo re ad ding the D NA se R NA se free w ater to the R NA pellet. 7.3.13 Add 500 :L of DNAs e R nase-free water to th e R NA pellet to d issolve it com pletely. Transfer the RNA in a 1.5 mL m icrotube.
Note :
The sam ple can be heated at 60/C in a hea t block and vortex to dissolve if nec ess ary.
Note:
The total RNA concentrate (oyster/viruses) can be stored at - 70/C or used imm ediately for purification and sub seq uen tly RT-PC R. 7.3.14 Transfer 400 :L of the total RNA to a 1.5 mL m icrotube and add 400:L 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.
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OPFLP-01 March 2010
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 :L of Dynabeads-oligo(dT) 25 to a 2.0 m L m icrotu be 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. Rem ove the m agnet slide from the m agnetic bead attractor. Note:
To prevent dam age to the m agnetic 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 :L 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 :L of binding buffer and add the 800 :L of total RNA (from step 7.3.14) to the 2.0 mL m icrotube containing the 100 :L 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 m inutes .
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 ma gnetic slide into the m agnetic bead attractor for 60 sec onds, then rem ove the binding buffer from the 2.0 m L m icrotube. R em ove the m agn etic slide .
Note:
If separatio n of m agnetic beads is incom plete prolong the incubation until the y separate properly 7.3.22 W ash the magnetic beads by adding 500:L of wash buffer to the 2.0 m icrotu be and m ix by inversion 5 times. Insert the magnetic slide into the magnetic bead attractor for 60 secon ds, then rem ove and disca rd the wash b uffer from the 2.0 m L m icrotube. Rem ove 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 m agnetic bead attractor. 7.3.23 Re pea t was h step (7.3 .22) 2 other times. 7.3.24 Rem ove last traces of final wash after microcentrifuging for 2 seconds and using the m agnetic slide into the m agnetic bead attractor. 7.3.25 Resuspend the magnetic beads with 25 :L of Rnase/DNase-free wate r an d elute PolyA viral RNA from beads at 90/C for 2 minutes in a heat block. 7.3.26 Separa te the heated solution from beads in the m agnetic 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 o f Rn ase inhibitor to the solution. 7.3.29 Store the 0.5 mL or 1.5 mL tube at – 70/C.
-77.4
7.5
Note:
Screen ing RT -PC R M eth od for N oro viru ses (i.e. de tection of o yster actin mRN A, No rov irus gen og rou ps 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 :L of the poly A RNA (7.3.29) to 20.0 :L of P CR reac tion m ixture (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 electroph ores is (7.5). If nec ess ary, the a m plicon s ca n be store d at 4/C for up to one week until analysis.
Agarose ge l electrop ho resis 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 susp ens ion).
7.5.2.
Cool agarose around 45 /C and a dd the ethidium brom ide co nce ntrate d so lution to have a final concentration of 0.5 :g/m L in the agarose gel (8 .8). Gently m ix while avoiding bubb le form ation.
The addition of EtBr to the gel is optional if the gel is submerged in EtBr solution after migration.
SAFET Y N OT E:
Note:
OPFLP-01 March 2010
ETHIDIUM BRO MIDE (EtBr) IS A POTENT MUT AGEN: 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 :L of tracking dye (loading buffer 10x concentrated ) with 25 :L of P CR prod uct.
7.5.5.
W hen the agaro se g el has set,re m ove the com b from the tray, place the tray with gel in the electrophoresis apparatus and fill reservoir with 0.5 X TBE buffe r to cover ge l. Gently pipet approximately 18 :L of samples (7.5.4) into the wells of the submerged gel. Pipet a s am ple of DN A m olecular size m ark er (e.g., 50 b p ladd er D NA ) in an e m pty 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 sam ple wells) and anode (+, red ) at the bottom (i.e., the end) of the gel. Ap ply approximately 135 volts to gel and run for about 30 minutes or until the OrangeG track ing dye has spre ad a distan ce o f app roximately 3/4 th of the length of the gel .
The voltage and time of migration can be changed according to the power supply used. 7.5.7
Rem ove gel from tray and visualize DNA bands by exposure to ultraviolet light (shortwave) using a transillum inator. Gels m ay be photog raphed on Polaroid™ 667 film to facilitate analysis and for record keeping purposes. Alternatively a digital processing system m ay be used.
-8Note:
OPFLP-01 March 2010
In the case whe re the EtBr has not been add ed d irectly to the gel, the gel must be rem ove from the tray and DNA stained by placing in ethidium bromide (EtBr) solution (0.5 :g/m L) for 30 to 45 m inutes (8.8). Rem ove the gel from EtB r us ing a gel scoop, rinse briefly with tap water,and visualize DNA bands by exp osu re to U V light.
SAFETY NOTE: 7.6
7.7
UV LIGHT CAN CAUSE EYE DAMAGE: WEAR SAFETY GOG GLES
Reading PCR R esults: 7.6.1
Th e am plicon (PC R produ ct) ge nera ted by the Ac tin-A/R prim ers, CO G1 F/R prim ers, COG 2F/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 reac tions.)
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 neg ative.
7.6.3
A spec ific band s hould appea r for the targeted norovirus positive control. Absen ce of a pos itive con trol ban d invalidates the tes t and the sam ples sho uld be re-analyzed . In cases where a positive reaction is observed for norovirus but no reaction is observed for the actin g ene m ay be indicative of a cros s co ntam ination betw een sam ples.
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 sam ples should be re-analysed using a fresh reagents.
7.6.5
Any test sa m ple sh owing a distinct band with the Norovirus prim ers, corre spo nding 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 foun d in the oyster.
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 :L) in 200 :L Reconditioning Solution and transfer suspension to a new RNase-free tube.
7.7.2
Incubate at 65/C in a drybath for 2 m inutes .
7.7.3
Pla ce tube in Dyn al
7.7.4
W ash 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
Plac e tube on the m agn et for a t least 30 se con ds a nd re m ove sup erna tant.
7.7.7
Re pea t steps 7.7.5 and 7.5.6 three tim es.
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
®
MP C-S for at lea st 30 sec ond s an d rem ove sup erna tant.
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OPFLP-01 March 2010
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: 8.
9.
Do not mix regenerated Dynabeads-oligo(dT) 25 with original stock suspension. 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 En viro nm ental M icrobiology 67:4152-4157.
8.2
Shieh, Y.C. Baric, R.S., W oods, J.W ., and Calci, K.R. 2003, Molecular surveillance of enterovirus and norwalk -like virus in oysters relocated to a m unicipal-sewa ge-im pacte d gulf estua ry. Applied and Environm ental M icrobiology. 69:7130-7136
8.3
Kageyama T, Kojima, S., Shinohara, M., Uchida, K., Fukushi, S.,Hoshino, F.B., Takeda, N. and Ka taya m a, K . 2003. Broadly re active and highly sensitive assay for No roviru ses based on rea ltime qu antitative reverse trans cription -PC R. Journ al of C linical Microbiology. 41:15 48-1557, V ol. 41, N o. 4
8.4
Dynal Biotech insert of Dynabeads-oligo(dT) 25 , printed 050 1, Rev. N o.: 004 available a t: www.invitrogen.com/content/sfs/manuals/Dynabeads%20Oligo.pdf
8.5
Anders on, A .D., H eryford, A.G ., Sarisky, J.P ., Higgins, C ., Monroe , S.S., B eard , R.S., New port, 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 am ong snowm obilers -W yom ing, 20 01. Journ al of Infectiou s Diseases . 187: 303-6.
8.6
Qiagen ®. 2002. Qiagen® OneStep RT-PCRKit Hanbook. For fast and efficient one-step RTPC R. 2800 Argentia R oad Unit 7 Mississauga O ntario L5N 8L2 . pp:10-12 . Available at: www1.q iagen.co m /literature/han dbo oks/PD F/PCR AndR everseTrans cription /KitsA ndE nzym es/R TPCR _OneStep/1020892HBRT PCR_05202.pdf
8.7
Sigma ®. 1999. Tech nical Bulletin MB-205 . Product Inform ation TRI-Re agent Produ ct T 9424 . (6 pag es) available at www.sigmaaldrich.com/sigma/bulletin/t9424bul.pdf
8.8
Sam brook, 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 Sm ith, C.S. 1983, Histological techniques for marine bivalve mollusks. NOAA Techncial Memorandum NMFS-F/NEC 25:97.
PC R PRIM ER S AND T EM PER ATU RE CY CL ING PROG RAM 9.1
PC R Prime rs Screening PCR: Oligonucleotide primers for the PCR are based on the most conserved region in the N orov irus geno m e. Kageyam a prime rs (8.3) for the detection of genogroup I Norovirus (85 bp fragm ent) : COG 1F - forward - 20 bases = 5'-C GY T GG AT G CG N T TY CAT GA- 3 ' COG 1R - reverse - 22 bases = 5'-C TT AG A C GC C AT CA T CA T TY A C -3' Kageyam a prime rs (8.3) for the detection of genogroup II Noro virus (98 bp fragm ent) : COG 2F - forward - 26 bases = 5'-C AR GAR BC N A TG TT Y AGR T GG AT G AG -3' COG 2R - reverse - 21 bases = 5'-T CG AC G CC A T CT TC A T TC A CA -3' Mon roe prime rs (8.5) for the detection of genogroup s I and II Norovirus (213 bp fragm ent) :
OPFLP-01 March 2010
- 10 431 432 433 434
-
forward forward reverse reverse
-
20 20 21 21
bases bases bases bases
= = = =
5'-T GG 5'-T GG 5'-G AA 5'-G AA
AC I AG R G GI CC Y AAY CA -3' AC I CG Y G GI CC Y AAY CA -3' YCT CA T CC A YCT GA A C AT -3' SC G CA T CC A R CG GAA CA T-3'
Actin house keeping gene target for oysters (8.2): Prime rs for the detection of oyster actin mRN A (257 bp fragm ent) : Actin-A - forward - 23 bases = 5'-T GG AA T CT G CYG GW A T CC AT G AA -3' Actin-R - reverse - 23 bases = 5'-CCG ATC CA G ACG GAG TAT T TC CT -3' Standard MixBase Definitions
Note :
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
Synthesis of oligonucleotide primers can usually be contracted out to a local university or, alte rnatively, m any biotec hnology firm s offe r a c ustom synthe sis servic e. If assistan ce is re quired in this matter, contact the authors. 9.2
Te mperatu re cycling pro gram for all Norov irus prim ers s pecific fo r geno gro up s I an d II and for the actin h ouse ke epin g ge ne target fo r oyste rs. The thermal cycler program should be set for the following sequence of cycling parameters :
Step No
Process
Tim e
Tempera ture
1
Reverse transcription
30 minutes
50/C
2
Initial PCR activation step
15 minutes
95/C
3
40 Cycles (a + b + c) 30 seconds 30 seconds 45 seconds
94/C 52/C 72/C
5 minutes
72/C
a. Melting b. Annealing c. Extension
4
Final elongation
Remarks
(HotStarTaq DNA Polymerase) Om niscript and Sensiscript Revers e Tran scriptases are inactivated and the cDNA template is denatured.
- 11 -
OPFLP-01 March 2010
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 recomm ended to have primers that have been purified by High Pressure/Performance Liquid Chromatography (HPLC)
10.
REAGENTS 10.1
Glycine bu ffer p H 9.5 7.50 g G lycine (m olecular biology grade). 17.5 5 g N aC l (m olecular biology gra de). Add distilled water to a volume of 800 m L , dissolve and adjust the pH to pH 9. Com plete to 1.0 L and autoclave, 121/C 15 m inutes or adjust at pH 9.5 and filter sterilize.
Note :
Th e final pH m ust be at 9.5 ± 0.1 afte r auto claving.
Note :
The glycine buffer pH 9.5 is stable for 9 months at 4 /C. 10.2
16 % polyethylene glycol (PEG) 80 g PE G 80 00 (m olecular biology grade). 15.3 4 g N aC l (m olecular biology gra de). Add distilled water to a volume of 500 m L dissolve and autoclave, 121/C 15 m inutes.
Note :
The PEG 16 % is stable for 9 months at 4 /C. 10.3
Binding Buffer 20 m M T ris-HCl, pH 7.5 (m olecular biology grade co m m ercially available). 1.0 M Lithium chloride (LiCl) (m olecular biology grade co m m ercially available). 2 m M E DT A (m olecular biology gra de c om m ercially 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 comm ercial solutions: 0.020 M of Tris-HCl, pH 7.5 * 50 mL = 0.1 M Tris-HCl * x mL x mL = 10 m L of 0.1 M Tris-HCl 1.0 M of LiCl * 50 mL = 8 M LiCl * x mL x mL = 6.25 m L of 8.0 M LiC l 0.002 M of EDTA * 50 mL = 0.5 M EDTA * x mL x mL = 0.200 mL of 0.5 M EDTA Com plete 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).
OPFLP-01 March 2010
- 12 10.4
Wash B uffer 10 m M T ris-HCl, pH 7.5 (m olecular biology grade co m m ercially available). 0.15 M lithium chloride (LiCl) (molecu lar biology grade com m ercially available). 1 m M ED TA (m olecular biology grade co m m ercially available). Exam ple of preparation of 50 mL of wash buffer from 0.1 M Tris-HCl, 8 M LiCl, 0.5 M EDT A and sterile distilled water (DNAse RNAse Free) from readily comm ercial 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 = 0.93 8 m L of 1.0 M LiC l 0.001 M of EDTA * 50 mL = 0.5 M EDTA * x mL x mL = 0.100 mL of 0.5 M EDTA Com plete 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
Qia gen O neStep R T-PC R K it 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 m aterials com ing into con tact w ith samples or RT -PCR rea gents 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 exp ose d to Noroviruses or Norovirus PCR products. To avoid any nonspecific amplifications, the mix should be prepared by putting all the reagents on ice or on a refrigerated rack.
Initial concentration
Stock solutions required for 20 reactions tubes using M onroe p rime rs
V olum e per tube
Final Concentration
---------
140.0 :L
7 :L
--------
5x
100.0 :L
5 :L
1x
10 m M'of each dNTP
20.0 :L
1 :L
400 :M
Prime r 431 - forward
10 :M'per primer
30.0 :L
1.5 :L
0.6 :M
Prime r 432 - forward
10 :M'per primer
30.0 :L
1.5 :L
0.6 :M
Primer 433 - reverse
10 :M'per primer
30.0 :L
1.5 :L
0.6 :M
Primer 434 - reverse
10 :M'per primer
30.0 :L
1.5 :L
0.6 :M
RT -PCR Com pon ents
RN ase-free wa ter 5x QIAGEN O neStep RT-PCR Buffer dN TP M ix
OPFLP-01 March 2010
- 13 20.0 :L
1 :L
--------
400 :L
20 :L
--------
20 :L
--------
--------
5 :L
--------
--------
Initial concentration
Stock solutions required for 20 reactions tubes using Kageyama primers for genogroups I or II and actin p rime rs
V olum e per tube
Final Concentration
---------
180.0 :L
9 :L
--------
5x
100.0 :L
5 :L
1x
10 m M'of each dNTP
20.0 :L
1 :L
400 :M
Primers COG 1F -forward or COG 2F - forward
10 :M'per primer
30.0 :L
1.5 :L
0.6 :M
Prime rs CO G1R - reverse or COG 2R - reverse
10 :M'per primer
30.0 :L
1.5 :L
0.6 :M
Primer Actin-A - forward
10 :M'per primer
10.0 :L
0.5 :L
0.2 :M
Primer Actin-R - reverse
10 :M'per primer
10.0 :L
0.5 :L
0.2 :M
---------
20.0 :L
1 :L
--------
400 :L
20 :L
--------
20 :L
--------
--------
5 :L
--------
--------
QIAGEN OneStep RTPCR Enzyme Mix
---------
T o tal V olu me Distribute per tube Tem plate
RT -PCR Com pon ents
RN ase-free wa ter 5x QIAGEN O neStep RT-PCR Buffer dN TP M ix
QIAGEN OneStep RTPCR Enzyme Mix T o tal V olu me Distribute per tube Tem plate
10.6
5 X Tris-Borate-EDTA - (TBE) buffer (or commercially available) Tris Base (m olecular biology grade). Boric Acid (m olecular biology grade). EDT A disodium (m olecular biology grade).
54.0 g 27.5 g 3.75 g
Add distilled w ater to a volum e of 8 00 m L, diss olve, com plete to 1.0 L. This buffer is used at a dilution (TB E 0.5 X B uffer) in distilled water.
1:10
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 , so dium salt
0.025 g
0.25 % (w/v)
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- 14 Glycerol Sterile distilled water (DNAse RNAse Free)
4 mL 6 mL
40%
(w/v)
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 mo lecular size marker (comm ercially available) Although many types of DNA size marker preparations are available from different suppliers, the 50 bp ladder DNA m arker provides a useful range of DNA fragment sizes and facilitates the "sizing" of PCR am plicons generated in this reaction.
10.9
Reconditioning solution for Dynabeads-oligo(dT)25 0.1 M N aOH (ACS grade or better). Example of preparation of 50 mL of stock solution of 1 M NaOH: W eight 2.0 g of NaO H and com plete with sterile distilled water (DN Ase R NAs e Free) to have 5 0 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 NaO H 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 m M T ris-HCl pH 7.5 (m olecular biology grade co m m ercially available). 20 m M ED TA (m olecular biology grade co m m ercially available). 0.1% T ween2 0 (m olecular biology grade co m m ercially available). 0.02% Sodium Azide (NaN 3) (ACS grade) Exam ple of preparation of 50 mL of storage buffer from 1.0 M Tris-HCl, 0.5 M EDT A, tween solution, S odium Azid e pow der an d sterile distilled wate r (D NAs e R NAs e F ree) from rea dily comm ercial solutions: 0.250 M of Tris-HCl, pH 7.5 * 50 mL = 1.0 M Tris-HCl * x mL x mL = 12.5 m L 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 :L of tween 20. Add 0.01 g of Sodium Azide (NaN 3) Add 33 .55 m L of sterile distilled water (DNA se RN Ase F ree). 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)
