Government of Canada Gouvernement du Canada

Government of Canada

Gouvernement du Canada

Laboratory Procedure

OPFLP-04 September 2007

HEALTH PRODUCTS AND FOOD BRANCH OTTAWA CONCENTRATION OF HEPATITIS A VIRUS AND ROTAVIRUS IN SPRING OR MINERAL BOTTLED WATER SAMPLES AND THEIR DETECTION BY THE REVERSE-TRANSCRIPTASE POLYMERASE CHAIN REACTION Carole Simard Peter Müller

Alain Houde Julie Brassard

Yvon-Louis Trottier

Validation and Technology Transfer Food Virology Unit

Food Research Development Centre

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

Agriculture and Agri-Food Canada 3600 Casavant Blvd W Saint-Hyacinthe, QC, Canada J2S 8E3

Health Products and Food Directorate Microbiology Laboratory Health Canada Quebec Region 1001, St-Laurent Blvd.W Longueuil, QC, Canada J4K 1C7

[email protected]

[email protected]

1.

[email protected]

APPLICATION This method is applicable for the concentration and detection of hepatitis A virus (HAV) and rotavirus from spring or mineral bottled water samples. This method will not differentiate between infectious and non-infectious viruses. It may not detect all strains of hepatitis A and rotaviruses found into the environment.

2.

DESCRIPTION This method has shown to produce excellent results using spring bottled water samples that were artificially inoculated with known amounts of HAV (strain HM175) and rotavirus (strain Wa). This method can also be adapted for the detection of others viruses when coupled with specific primers (e.g. hepatitis E virus (HEV), Norovirus genogroups I and II, Adenovirus types 40/41 or other viruses). This procedure allows the filtration of water samples while eliminating potential inhibitors (8.4). This technique is simple, fast and economical and can be easily transferred to diagnostic laboratories.

______________________ Published on the Food Directorate’ (Health Canada) Website at http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index_e.html

3.

PRINCIPLE Samples of bottled water that are potentially contaminated by hepatitis A virus and/or rotavirus (or other viruses) are filtered through a positively charged membrane. Elution of viruses attached to the membranes is achieved with a tryptose phosphate broth-glycine buffer. The eluates are further concentrated using Microsep 100TM or CentriconTM columns. The RNA is then extracted using the Qiagen RNeasy kit followed by an evaporation step with a SpeedVac instrument. Finally, the concentrated RNA is then subjected to a RT-polymerase chain reaction (RT-PCR) procedure which amplifies a specific fragment depending on the primers used (e.g. HAV, rotavirus, HEV, Norovirus genogroups I and II, Adenovirus types 40/41 or other viruses).

4.

DEFINITION OF TERMS See Appendix A of Volume 3.

5.

COLLECTION OF SAMPLES See Appendix B of Volume 3.

Note:

6.

The number of samples may have to be determined on the basis of specific client needs (e.g. Data collection or surveys) or investigational purposes (e.g. Outbreaks).

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".

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 :L, 10 to 100 :L, and 100 to 1000 :L 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, 56/C and 98/C.

Note:

It is the responsibility of each laboratory to ensure that the heat blocks are maintained at the recommended temperatures. Where 37/C is recommended the heat blocks may be at 37/C +/1/C. For all other temperatures it may be +/- 2/C. For freezers, it may be at -20 or -70 +/- 5/C.

8)

12 mL tubes.

9)

Vortex mixer.

10)

Microfuge tube - 1.5 mL.

11)

SORVALL™ model RC-5B Plus refrigerated centrifuge with SM-24 rotor or equivalent.

12)

47 mm Filtration kit (VWR cat. Number 26316-708) no substitution.

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OPFLP-04 Draft September 2006

13)

47 mm diameter filters, Zeta plus™ 60S (WEIR Canada cat. Number B0204-60S) no substitution.

14)

Orbital Shaker, Orbit 300 Model ™150 or equivalent.

15)

15 mL centrifugation tubes.

16)

KNF NewBurger™ Vacuum pump or equivalent.

17)

SpeedVac™, Savant Instrument model DNA-110-120 or equivalent.

18)

Tweezers.

19)

Microfuge.

20)

Tubes for PCR - thin wall 0.2 mL.

21)

Timer.

22)

Container for ice.

23)

Magnetic stirrer.

24)

Freezers capable of maintaining - 20 /C and - 70/C.

25)

Qiagen ™OneStep RT-PCR Kit (product numbers 210210 or 210212, no substitution). (8.5)

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 or equivalent (re. For tracking dye in loading buffer in 10.4 ). Glycerol (Molecular biology grade - for loading buffer in 10.4) DNA molecular weight standard ladder 100 bp or equivalent. Ethidium bromide (molecular biology grade). Tris[hydroxymethyl]aminomethane (molecular biology grade). Boric Acid (molecular biology grade). Ethylene Diamine Tetra Acetic acid (EDTA) disodium salt (molecular biology grade) Water DNAse RNAse free (molecular biology grade). 10 % SDS Filtered Sigma cat. Number L4522 or equivalent (molecular biology grade). TPBG buffer pH 9.0 (2.9 % Tryptose Phosphate Broth 6 % Glycine) (See section 10.1 for preparation). Hydrochloric acid (HCl) 1N (ACS grade or better). Sodium Hydroxide (NaOH) 10 N (ACS grade or better). Pure Ethanol (molecular biology grade). 14.3 M ß- mercaptoethanol (molecular biology grade). Loading Buffer (10.4)

27)

RNeasy™ Mini Kit (Qiagen Cat. Number 74103, 74104 or 74106) no substitution. (8.6)

28)

20 mg / mL Proteinase K (Invitrogen Life Technologies cat. Number 25530-049) or equivalent.

29)

Universal pH indicator test strips, VWR cat. Number 60775-000 or equivalent.

30)

SORVALL™ model Legend RT refrigerated centrifuge with number 3332 rotor or equivalent.

31)

SH-Prot-1 and SH-Prot-A for the detection of HAV (8.8) (See section 9.1.1 for DNA sequence).

32)

Jean primer (Rota-1) / Gouvea primer (End 9) for the detection of rotavirus (See section 9.1.2 for DNA sequence).

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7.

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33)

65 mm Petri Dish.

34)

1 L erlenmeyer with 6 mm side arm.

35)

Microsep 100 K™ columns with a Molecular Weight cutoff of 100 000 daltons (Pall Life Sciences cat Number OD100C41 or OD100C46) or equivalent (e.g. Centricon, Amicon). (8.7)

36)

Tryptose Phosphate Broth (Oxoid #CM283 or BBL-DIFCO #DF0060-17-8 or equivalent)

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. In the meantime, keep samples at 4/C.

7.2 Note :

Preparation for Analysis It is important to ensure that water samples are at room temperature before proceeding. 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 (e.g. HAV or rotavirus).

7.3

Virus concentration, RNA extraction and concentration from spring water samples

SAFETY NOTE : ADDITIONAL PRECAUTIONS MUST BE IMPLEMENTED WHEN WORKING WITH BOTTLED WATER SAMPLES THAT ARE POTENTIALLY CONTAMINATED WITH HAV, ROTAVIRUS OR OTHER VIRUSES. VIRUS CONCENTRATION AND EXTRACTION ARE CARRIED OUT UNDER A BIOLOGICAL CONTAINMENT HOOD WITH A MASK, SLEEVE PROTECTORS AND DOUBLE NITRILE GLOVES.

7.3.1 Filtration and elution of viruses using a positively charged membrane. 7.3.1.1

Install the 47 mm diameter Zeta plus 60S filter on top of the filter support using sterile tweezers.

Note :

It is important to place the smooth surface of the Zeta filter facing downward in order to expose the positively charge membrane surface to the water sample.

Note :

All glassware must be thoroughly clean and sterile prior to use.

Note :

Use a different funnel for each water sample to analyse. 7.3.1.2

Center the Zeta filter and position the graduated funnel on top of the filter.

7.3.1.3

Use the stainless steel support filter holder (clamp) to make a water tight seal.

7.3.1.4

Connect the 1000 mL Erlenmeyer to a vacuum pump.

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Note:

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The filtration step is conducted inside a biological containment hood to limit the risk of exposure to aerosols and to work in a sterile environment. 7.3.1.5

Pour approximately 20 mL of sterile water (DNAse RNAse free) to hydrate the filter and to verify the effectiveness of the water tight seal.

7.3.1.6

Start the pump and adjust the flow of water to a slow trickle.

7.3.1.7

Pour the 100 mL water sample inside the glass funnel and stop the vacuum pump once the water sample is completely filtered.

7.3.1.8

Remove the stainless steel support filter holder (clamp) and carefully withdraw the graduated funnel on top of the Zeta filter.

7.3.1.9

Remove the Zeta filter from the filter support using sterile tweezers and place the filter inside a sterile 65 mm Petri dish.

7.3.1.10 Add 6 mL of TPBG buffer pH 9.0 (2.9 % Tryptose Phosphate Broth 6 % Glycine) to the Zeta filter. Cover the Petri dish using its lid. Note:

When placing the filter into the petri dish, please ensure that the filter is placed upside down (inverted). 7.3.1.11 Place the Petri dish on the orbital shaker and adjust the speed of rotation to 30 rpm. Incubate the orbiting Petri dish for 15 minutes at room temperature (23 /C ± 3 /C). 7.3.1.12 Stop the orbital shaker and remove the eluate (~5 mL) containing the viral particles and transfer into a 12 mL tube. 7.3.1.13 Adjust the pH of the eluate (~5 mL) to 7.0 -7.4 with (around 250:L for 5 mL of eluate) 1N HCl solution and pH indicator test strips.

Note:

The eluate can be stored at - 70/C as a stopping point.

7.3.2 Concentration of virus particles using Microsep 100 K columns with a molecular weight cutoff of 100 000 daltons. 7.3.2.1

Transfer the remaining eluate (~5 mL) into equal volumes in two separate Microsep columns.

7.3.2.2

Centrifuge 60 minutes at 5000 x g.

7.3.2.3

Rinse gently the surface with the remaining volume. Pool the paired concentrate of each sample together (~150 :L).

7.3.2.4

Transfer into a 1.5 mL microfuge tube and measure the total volume of eluate of each sample.

7.3.2.5

Complete the volume to 150 :L with DNase RNase free water if necessary.

7.3.3 Extraction of RNA using Qiagen Rneasy kit. 7.3.3.1

For each sample, add 15 :L of SDS 10% to achieve a final concentration of 1% SDS.

7.3.3.2

Add 0.75 :L of Proteinase K 20 mg/ mL per sample to have a final concentration of 100 :g/mL of Proteinase K.

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7.3.3.3

Incubate the 1.5 mL microfuge tube one hour at 37 /C in a heat block.

7.3.3.4

Add 450 :L of RLT-ß- mercaptoethanol solution per sample.

SAFETY NOTE: ß- MERCAPTOETHANOL IS TOXIC, DISPENSE IN A FUME HOOD AND WEAR APPROPRIATE CLOTHING. Note:

Note:

Add 10 :L of ß- mercaptoethanol per 1 mL Buffer RLT. Buffer RLT is stable for 1 month after the addition of ß- mercaptoethanol. Store at room temperature.

7.3.3.5

Vortex the samples 15 seconds.

7.3.3.6

Heat the samples 2 minutes at 56 /C in a heat block.

7.3.3.7

Incubate an additional 5 minutes at room temperature (23 /C ± 3 /C).

7.3.3.8

Add 500 :L of pure ethanol per sample and mix well by pipetting.

7.3.3.9

Apply up to 700 :L of the sample to a supplied RNeasy column. Close the tube gently and centrifuge for 15 seconds at 8000 x g.

Include any precipitate that may have formed to the column. 7.3.3.10 Repeat the previous step until all of the solution has run through the RNeasy column. 7.3.3.11 Add 700 :L of Buffer RW1 through the RNeasy column. Close the tube gently and centrifuge 15 seconds at 8000 x g. 7.3.3.12 Transfer the RNeasy column into a new 2 mL collection tube. Add 500 :L RPE onto the RNeasy column. Close the tube gently and centrifuge for 15 seconds at 8000 x g.

Note:

Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to buffer before use. 7.3.3.13 Add another 500 :L of Buffer RPE to the RNeasy column. Close the tube gently and centrifuge for 2 minutes at 8000 x g. 7.3.3.14 Transfer the RNeasy column into a new 2 mL collection tube and centrifuge for 1 minute at 10 000 x g ensure that the column is dry. 7.3.3.15 Transfer the column into a 1.5 mL microfuge tube and add 30 :L of DNase RNase-free water supplied in the kit directly to the filter of the RNeasy column and close the tube gently. Centrifuge for 1 minute at 8000 x g. 7.3.3.16 Add another 20 :L of DNase RNase-free water directly to the filter of the RNeasy column and close the tube gently. Centrifuge for 1 minute at 8000 x g. 7.3.3.17 A final volume of approximately 50 :L is obtained. 7.3.4 RNA concentration using a SpeedVac instrument. 7.3.4.1

Place the 1.5 mL microfuge tubes with open lids into the SpeedVac rotor to permit evaporation of water.

7.3.4.2

Close the lid, power on the SpeedVac instrument and select the High speed function and start the cycle using the Concentrator switch.

7.3.4.3

Treat the samples for up to 30 minutes on the SpeedVac or for the length of time required to remove the excess solvent up to a volume of around 10 :L.

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Note:

7.4

-


Verify the residual volume of each sample every 15 minutes and close the lid when the targeted volume is obtained. 7.3.4.4

Complete the volume of each sample to 10 :L if necessary using DNase Rnase free water.

7.3.4.5

Proceed to the RT-PCR screening test.

Screening RT-PCR Method for HAV or rotavirus 7.4.1 Screen the concentrated RNA of each water sample with the one set of HAV primers (9.1.1) and one set of rotavirus primers (9.1.2) using the OneStep RT-PCR kit from Qiagen (one set of primers per reaction as per 10.2 , no multiplexing). 7.4.2 Incubate the concentrated RNA at 98/C for 5 minutes followed by a rapid chilling on ice.

Note:

This step is required only for rotavirus because of the double-stranded RNA nature of the virus. 7.4.3 Add 2.0 :L of the concentrated RNA (7.4.2) to 18.0 :L of PCR per reaction mixture (10.2). 7.4.4 Insert microfuge tubes in a thermal cycler and proceed with RT-PCR amplification according to the program described under 9.2. 7.4.5 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 until analysis.

7.5

Agarose gel electrophoresis 7.5.1 Prepare a 1.5% (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 2.5 :L of Ethidium Bromide Solution (EtBr 10mg/mL). 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. Handle in a fume hood.

7.5.3 Pour into a gel tray having both ends sealed with tape. 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 2.5 :L of tracking dye ( loading buffer 10 x concentrated ) with 25 :L of PCR product. 7.5.5 When the agarose gel has set, remove the comb and tape from the tray, place the tray with gel in the electrophoresis apparatus and fill the reservoir with 0.5 X TBE buffer to cover gel with buffer to a depth of 4 mm. Gently pipet approximately 18 :L of samples (7.5.4) into the wells of the submerged gel. Pipet a sample of DNA molecular size marker (e.g., 100 bp ladder DNA) into an empty well. Include positive, negative and reagent controls.

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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 tracking dye has spread a distance of approximately two-thirds the length of the gel. Note:

The voltage and time of migration can be adapted in accordance with 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 removed from the tray and DNA stained by placing in ethidium bromide (EtBr) solution (10 g/mL) for 15 min. 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 amplicons (PCR product) generated by the HAV primers and rotavirus primers are double stranded DNA fragments of 172 bp and 268 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., 100 bp ladder DNA) run on the same gel. 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 (non-specific amplification products) is not considered as a presumptive positive. 7.6.3 A specific band should appear for the targeted positive control. Absence of a positive control band invalidates the test and the samples should be re-analyzed. 7.6.4 Any band corresponding to the positive control occurring in the negative or reagent control with either the HAV or rotavirus 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 new reagent batch. 7.6.5 Any test sample showing a distinct band with the HAV or rotavirus 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 HAV or rotavirus found in the water 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.

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8.

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REFERENCES 8.1

Brassard, J., Seyer, K., Houde, A., Simard, C. and Trottier, Y.-L. 2005. Concentration and detection of hepatitis A virus and rotavirus in spring water samples by reverse transcription-PCR. Journal Virological Methods, Volume 123:2, pages 163-9

8.2

Gouvea, V., Glass, R.I., Woods, P., et al. 1990. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. Journal of Clinical Microbiology, Volume 28, pages 276-82.

8.3

Jean, J., Blais, B., Darveau, A. and Fliss, I. 2002. Simultaneous detection and identification of hepatitis A virus and rotavirus by multiplex nucleic acid sequence-based amplification (NASBA) and microtiter plate hybridization system. Journal Virological Methods, Volume 105, pages 123-32.

8.4

Queiroz, A.P.S.,Santos, F.M., Sassaroli, A., Hársi, C.M., Monezi,T .A. and Mehnert, D.U. 2001. Electropositive filter membrane as an alternative for the elimination of PCR inhibitors from sewage and water samples. Applied and Environnemental Microbiology, Volume 67, pages 4614-8.

8.5

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: http://www1.qiagen.com/literature/handbooks/PDF/PCRAndReverseTranscription/KitsAndEnz ymes/RTPCR_OneStep/1020892HBRTPCR_05202.pdf (Accessed on May 29th 2006)

8.6

Qiagen® RNeasy Mini Hanbook. Third Edition . June 2001. Argentia Road Unit 7 Mississauga Ontario L5N 8L2. pp:10-12. Available at: http://www1.qiagen.com/literature/handbooks/PDF/RNAStabilizationAndPurification/FromAni malAndPlantTissuesBacteriaYeastAndFungi/RNY_Mini/1016272HBRNY_062001WW.pdf (Accessed on May 29th 2006)

8.7

Microsep™ & Microsep MF Centrifugal Devices. 2005 Pall Corporation. Available at: http://www.pall.com/variants/pdf/pdf/20048.pdf (Accessed on May 29th 2006)

8.8

Guévremont, E., Brassard, J., Houde, A., Simard, C. and Trottier, Y.-L. 2006. Development of an extraction and concentration procedure for the detection of hepatitis A virus and norovirus from green onions by RT-PCR. Journal of Virological methods, Volume 134:1-2, pages130-5.

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9.


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PCR PRIMERS AND TEMPERATURE CYCLING PROGRAM 9.1

PCR Primers 9.1.1 Screening RT-PCR for HAV : The primers SH-Prot-A and SH-Prot-1 were designed based upon the protease coding region 2 A of HAV and gave an amplification product of 172 bp (8.8). Guévremont primers for the detection of HAV (172 bp fragment) : SH-Prot-A

- forward - 20 bases (3378-3397)

5'- ATG GAT GCT GGR GTT CTT AC -3' SH-Prot-1

- reverse - 22 bases (3529-3550)

5'- ART TGG CAG CAA TTT CTT CAA G -3'

Note :

Numbers refer to the corresponding nucleotide positions of reference hepatitis A virus strain HM-175 wild type (GenBank accession no. NC 001489)

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

9.1.2 Screening RT-PCR for rotavirus: Oligonucleotide primers for the RT-PCR are based upon the highly conserved region in gene segment 9 which encodes vp7 of rotavirus (8.2 and 8.3). Jean and Gouvea primers for the detection of rotavirus (268 bp fragment) :

Note:

Rota-1 - forward - 21 bases =

5'-GTA AGA AAT TAG GTC CAA GAG-3'

End 9 - reverse - 27 bases =

5'-GGT CAC ATC ATA CAA TTC TAA TCT AAG-3'

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.

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Note:


-

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 HAV and rotavirus primers The thermal cycler program should be set for the following sequence of cycling parameters:

Step No

Process

Time

Temperature

1

Reverse transcription

30 minutes

50/C

2

Initial PCR activation step

15 minutes

95/C

3

35 Cycles (a + b + c) a. Melting

45 seconds

b. Annealing

45 seconds

Remarks

(HotStart Taq DNA Polymerase) Omniscript and Sensiscript Reverse Transcriptases are inactivated and the cDNA template is denatured.

94/C 53/C (HAV) 43/C (Rotavirus)

c. Extension

60 seconds 72/C

4

Final elongation

5 minutes (HAV)

72/C

5 minutes (Rotavirus) indefinite (Stand by)

Note:

10.

4/C

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.

REAGENTS 10.1 TPBG buffer pH 9.0 (2.9 % Tryptose Phosphate Broth 6 % Glycine) 2.9 g Tryptose Phosphate Broth.

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g Glycine (analytical grade or better).

Add 80 mL of DNase RNase-free water and adjust the pH to 9.5 with a NaOH 10N solution. Complete to 100 mL and filter sterilize.

Note:

The TPBG buffer should be stable for 9 months at 4 /C, discard if cloudy.

10.2 Qiagen OneStep RT-PCR Kit (8.5) 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 HAV or rotavirus 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. Also, for reducing the cost of reagents, 20 :L of RT-PCR mix dispensed in 200 :L tubes are used.

RT-PCR Components

Initial concentration

Stock solutions required for 20 reaction tubes using HAV or rotavirus primers

Volume per

Final Concentrati on

tube

RNase-free water

---------

200.0 :L

10 :L

--------

5x QIAGEN OneStep RT-PCR Buffer

5x

100.0 :L

5 :L

1x

10 mM'of each dNTP

20.0 :L

1.0 :L

400 :M

10 :M'per primer

30.0 :L

1.5 :L

0.6 :M

10 :M'per primer

30.0 :L

1.5 :L

0.6 :M

dNTP Mix

Primer SH-Prot-1 forward or Rota-1 forward Primer SH-Prot-2 reverse or End 9 - reverse

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20.0 :L

1.0 :L

--------

Total Volume

400 :L

20 :L

--------

Distribute per tube

20 :L

--------

--------

Template

5 :L

--------

--------

QIAGEN OneStep RT-PCR Enzyme Mix

---------

10.3 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. 10.4 Tracking dye / Loading Buffer or equivalent. 10X Orange G, sodium salt

0.025 g

0.25 % (w/v)

Glycerol

4 mL

40%

Sterile distilled water (DNAse RNAse Free)

6 mL

(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.

Note:

Store the working solution at 4/C.

10.5 DNA molecular size marker (commercially available) Although many types of DNA size marker preparations are available from different suppliers, the 100 bp ladder DNA marker provides a useful range of DNA fragment sizes and facilitates the "sizing" of PCR amplicons generated in this reaction.