Rotavirus Detection and Typing




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Appendix ii

Rotavirus Detection and Typing



Nucleic acid extraction and reverse transcription

Virus Detection by PCR

Rotavirus VP7, VP4, VP6 and NSP4 genotyping


Version 4
01.10.2009

Contents
1. Specimen Storage and Preparation of 10% Faecal Suspension 3

2. RNA Extraction 4

2.1 Manual ‘Boom’ Extraction 4 2.2 Automated extraction: QIAxtractor (VX extraction kit, QIAGEN) 5

3. Random Priming RT 9

4. Rotavirus VP6-specific PCR 10

5. Rotavirus VP6-specific real-time PCR (Taqman) 11

6. Rotavirus NSP4-specific PCR 12

7. Rotavirus genotyping PCRs 13

7.1 G-Typing consensus PCR (VP7) 13

7.2 G-Typing Multiplex PCR 14

7.3 VP7 semi-nested consensus PCR (sequence typing) 15

7.4 P-Typing consensus PCR (VP4) 16

7.5 P-Typing multiplex PCR 17

8. Oligonucleotide primer and probe sequences 18

9. RNA extraction reagents 22

10. Disposal of toxic waste 24

11. Agarose-gel electrophoresis 25

NOTE: All risk assessments and COSHH assessments associated with the following procedures should be performed locally



1. Specimen Storage and Preparation of 10% Faecal Suspension


  • Upon receipt, the specimens should be stored at 4oC.

  • Coat with a layer of glycerol if the specimens are not going to be processed within a week.

  • Prepare 10-20% faecal suspensions in balanced salt solution (BSS) (Minimum Essential Medium or Medium 199) in 2ml screw cap tubes (Sarstedt).

  • Add 200ul of liquid sample to 2 ml of BSS or a bacteriological loop full (the size of a garden pea) from semi-solid samples.

  • Store the 10% faecal suspension at 4oC until processing.

  • Once all investigations have been carried out the 10% suspensions can be stored at -70 oC. The original faecal sample should be stored at 4oC under a layer of glycerol.



Avoid freezing and thawing the specimens repeatedly


2. RNA extraction
2.1 Guanidinium isothiocyanate (GTC)/ silica gel extraction of nucleic acid

The 'Boom' method [Boom et al., 1990 J Clin Microbiol].





  1. Add 200ul of faecal suspension to 1ml of lysis buffer-L6 (see section 8) buffer and 20ul of size fractionated silica (see section 8) in a 1.5ml screw-capped microcentrifuge tube. RNase-free distilled water is included in each run to act as a negative control. Cell-culture grown SA11 or RRV should be used as a positive control1.

2. Vortex for 10 sec and incubate at RT for 15 min.

3. Pellet by centrifugation (microcentrifuge) for 15 sec (13,000rpm), discard the supernatant. (Collect the supernatants for disposal of toxic waste, see Disposal of toxic waste; see section 9).

4. Wash the pellet with 1ml of lysis buffer L2 (see section 8), mix well by vortexing and pellet by centrifugation as before. Repeat with L2 buffer then similarly wash twice with 1ml 70% ethanol and 1ml acetone once2 (store the wash fluids for disposal).

5. After removal of the acetone (perform carefully as pellets may become dislodged) centrifuge and place tube with lid open at 56oC in a dry heating block for 5 minutes.

6. Add 49ul of RNase-free distilled water and 1ul of RNasin, vortex and incubate at 56oC for 15 min to elute the nucleic acid from the silica.

7. Pellet by centrifugation at 13,000rpm for 4 min and extract the supernatant (avoid disturbing the silica. Recentrifuge if silica becomes resuspended). This can be stored in a new microfuge tube at 4oC for 24h or -70oC for longer.


2.2 QIAxtractor from QIAGEN™

Please refer to manufacturer’s instructions for instrument set-up and calibration


VXB (Binding Buffer)
VXB Binding Buffer 100ml Store at room temperature

VXB Binding Additive 1.14g Store at 4°C


If a whole bottle of VXB will be used up in <1month, add all the additive to the VXB bottle.
VXL (Lysis Buffer)

Reconstitute Carrier RNA in 400ul Elution Buffer

VXL Lysis Buffer 10ml Store at room temperature

VX Proteinase K 1.1ml Store at 4°C

Carrier RNA 210ul Store at 4°C





  1. Prepare Lysis Plate by adding 200ul of faecal suspension (see section 1.) to each well. RNase-free distilled water is included in each run to act as a negative control. Cell-culture grown SA11 or RRV should be used as a positive control3. The wells of whole columns need to be used, so if there are insufficient samples, then 200 ul of water should be added to the remaining wells in the column.

If the whole of the plate is NOT used seal unused columns on the capture plate with an adhesive plastic plate seal

  1. Turn on the QIAxtractor followed by the computer.

  2. Select and double-click on the protocol required.

  3. A map of the QIAxtractor platform layout is displayed on the screen.

  4. The nucleic acid Capture Plate is positioned in area A1.

    1. Remove the Waste Sink from the left hand side of area A1.

    2. On the bench, turn the Sink upside down and position over Silicone Mat.

    3. Line up the mat and Sink and apply downward pressure to insert the Silicone Mat.

    4. Smear a small amount of silicone grease on the Sink O ring.

    5. Insert the Sink into A1 with the nozzle inserted into the vacuum chamber.

    6. Place the Transfer Carriage on top of the Silicone Mat and Waste Sink with the nozzles aligned with the holes in the Silicone Mat (do not use excessive pressure).

  5. Place the Elution Riser block into the right hand side of the vacuum chamber and position the Elution Plate on top of the Riser.

  6. On the platform layout, the reagents required, the positions of the reagent tubs and the volume of the reagents required will be highlighted in pink (areas R1 and C1).

    1. If less than 96 samples in 12 columns are to be extracted, then edit the programme to set the appropriate number of columns.

      1. Open the wizard and press [Clear All]

      2. Click on the required columns, click [Jump to End], then click [Finish].

    2. Fill the reagent tubs with the solutions displayed and the required volumes shown on the screen. The tubs have graduated measurements on the side for ease of volume measurement. Always ensure slightly more reagent (~5ml) is added to each tub to avoid running out during the run. The Lysis Buffer is already made up in excess.

a. The reagent tubs must have their lids in place before the run starts

b. Reagent tubs are reusable. Label the tubs, and rinse with water before each use.

  1. The Tip Racks are placed on positions B2 and C2 using the mouse and highlight the tips that are available for use.

  2. Check that the lids are removed the Tip Racks and Elution Plate, check that the cover has been removed from the waste-tip shute and the waste tip collector is in place.

  3. Ensure the elution volume is set to 80ul.

  4. Click on [Start] then tick the checklist and click [OK] to begin the run.

    1. The QIAxtractor pauses after the vacuum step has drawn the sample through the Capture Plate. Check that the wells are not blocked. If clear, click on [Continue].

    2. Blocked well can be cleared by puncturing the membrane with a needle. Use a clean needle for each blocked membrane, record the plate positions and then click on [Continue].

  5. When the run has completed, place the lid on the Elution Plate (containing extracted RNA), remove from the robot and place on ice. Follow RT protocol as described (see section 3).

The RT and subsequent PCRs can be performed in 96 well plates.

  1. After the run has completed, decontamination needs to be performed before the start of the next run.

    1. Remove the Capture Plate, Silicone Mat and empty tip racks and discard.

    2. Relid unsued tips and remove from the platform.

    3. Remove reagent tubs, discard any remaining liquid to waste, wash in tap water, rinse in distilled water and leave to dry.

    4. Remove the Waste Sink and Elution Riser, wash in tap water, rinse in distilled water and leave to dry.

DO NOT heat or autoclave any of the washed components.

    1. Remove the Transfer Carriage, wipe the Transfer Carriage and the platform with nucleic acid decontamination wipes followed by alcohol wipes.

    2. Add 50ml of water to the drain and purge by clicking on the ‘bucket’ icon and follow the instructions.

    3. Dry the vacuum chamber with paper towel, wipe with nucleic acid decontamination wipes followed by alcohol wipes.

    4. Cover the tip shute with the metal cover provided.

    5. Run the UV decontamination cycle by pressing the ‘bulb’ icon and follow the instructions.

    6. When the UV cycle is complete, replace all the washed plastic reagent tubs, the Elution Rise, Transfer Carriage and Waste Sink back on the platform, ready for the next run and repeat the UV decontamination cycle.

    7. When the UV cycle is complete, remove the tip shute cover, switch off the robot and then shut-down the computer.



3. Random Priming RT


  1. Prepare RT mix for N + 2 (N = No. of tubes in test)
  2. 1x mix

        1. 10X buffer II (Invitrogen4) 7.0ul


        2. 50mM MgCl2 7.0ul

        3. Random primers5 1.0ul

        4. dNTPs (10mM) 2.0ul
        5. M-MLV (200U/ul) Invitrogen 2.0ul


        6. RNase-free H2O 11.0ul

30.0ul


  1. Transfer 40ul of extracted nucleic acid to a PCR tube.

  2. Denature the dsRNA at 97oC for 5 min.

  3. Chill the tubes on ice for 2 min.

  4. Add 30ul to each tube containing the extracted RNA.

  5. Incubate the tubes at 37oC for 1h.

  6. Incubate the tubes at 95oC for 5 min.

  7. Chill the tubes on ice for 2 min.

  8. The total volume should be 70ul.

  9. The cDNA can be used directly in the PCR, and stored at -20oC for further use.

4. Rotavirus VP6-specific PCR6

1. Prepare PCR mix for N + 2 (N = No. of tubes in test)

1x mix

10X buffer II (Invitrogen) 4.5ul



50mM MgCl2 2.0ul

dNTPs (10mM) 1.0ul

Taq Polymerase (5U/ul) (Invitrogen) 0.2ul

primer VP6-F7 (20pmoles/ul) 1.0ul

primer VP6-R (20pmoles/ul) 1.0ul

RNase-free H2O 35.3ul

45.0ul
2. Add 45ul of PCR mix to each PCR tube.

3. Add 5ul cDNA (from the RT reaction: section 3).

4. Briefly spin in microcentrifuge (pulse for 5 sec).

5. Transfer tubes to the PCR machine.

6. Add tubes to thermocycler and cycle at the following temperatures for the

following times:



94oC 2min X1

94oC 1min

55oC 1min X35

72oC 1 min
72oC 7min X1
15oC Hold

5. Rotavirus VP6-specific real-time PCR (Taqman)


  1. Prepare PCR mix for N + 2 (N = No. of tubes in test)

1x mix

Taqman MasterMix 2x (Invitrogen) 12.5ul

ROX (Invitrogen) 0.5ul

primer VP6-F8 (20pmoles/ul) 1.0ul

primer VP6-R (20pmoles/ul) 1.0ul

VP6probe (20uM) 0.2ul

RNase-free H2O 7.8 ul

23.0ul



  1. Add 23ul of Taqman mix to each well of a 96 well plate.

  2. Add 2ul cDNA (from the RT reaction: section 3).

  3. Ensure there are no bubbles in the wells and seal the plate with the optical covers.

  4. Briefly spin in plate centrifuge (pulse for 5 sec).

  5. Transfer plate to and ABI PRISM 7500 SDS Taqman machine.

  6. Cycle at the following temperatures for the following times:



50oC 2min X1

95oC 2min X1

95oC 15sec

60 oC 1min X35

All the wells should be assayed using the dye and quencher of the probe



6. Rotavirus NSP4-specific PCR9

1. Prepare PCR mix for N + 2 (N = No. of tubes in test)

1x mix

10X buffer II (Invitrogen) 4.5ul



50mM MgCl2 2.0ul

dNTPs (10mM) 1.0ul

Taq Polymerase (5U/ul) (Invitrogen) 0.2ul

primer NSP4-F10 (20pmoles/ul) 1.0ul

primer NSP4-R (20pmoles/ul) 1.0ul

RNase-free H2O 35.3ul

45.0ul
2. Add 45ul of PCR mix to each PCR tube

3. Add 5ul cDNA (from the RT reaction: section 3)

4. Briefly spin in microcentrifuge (pulse for 5sec).

5. Transfer tubes to the PCR machine.

6. Add tubes to thermocycler and cycle at the following temperatures for the

following times:



94oC 2min X1

94oC 1min

48oC 1min X35

72oC 1 min
72oC 7min X1
15oC Hold

7. Rotavirus genotyping PCRs

7.1. G-Typing consensus PCR (VP7)
1. Prepare 1st round PCR mix for N + 2 (N = No. of tubes in test)

1x mix


10X buffer II (Invitrogen) 4.5ul

50mM MgCl2 2.0ul

dNTPs (10mM) 1.0ul

Taq Polymerase (5U/ul) (Invitrogen) 0.2ul

primer VP7-F11 (20pmoles/ul) 1.0ul

primer VP7-R (20pmoles/ul) 1.0ul

RNase-free H2O 35.3ul

45.0ul


2. Add 45ul of PCR mix to each PCR tube.

3. Add 5ul cDNA (from the RT reaction).

4. Briefly spin in microcentrifuge (pulse for 5sec).

5. Transfer tubes to the PCR machine.

6. Add tubes to thermocycler and cycle at the following temperatures for the

following times:



94oC 2min X1

94oC 1min

52oC 1min X35

72oC 1min
72oC 7min X1
15oC Hold
7.2. G-Typing multiplex PCR

1. Prepare 2nd round PCR mix for N + 2 (N = No. of tubes in test)

1x mix

10X buffer II (Invitrogen) 4.8ul



50mM MgCl2 2.5ul

dNTPs (10mM) 1.0ul

Taq Polymerase (5U/ul)(Invitrogen) 0.2ul

primer VP7-R12 (20pmoles/ul) 1.0ul

primer G1 (20pmoles/ul) 1.0ul

primer G2 (20pmoles/ul) 1.0ul

primer G313 (20pmoles/ul) 1.0ul

primer G4 (20pmoles/ul) 1.0ul

primer G815 (20pmoles/ul) 1.0ul

primer G914 (20pmoles/ul) 1.0ul

primer G1015 (20pmoles/ul) 1.0ul

primer G1216,15 (20pmoles/ul) 1.0ul

RNase-free H2O 30.5ul


  1. Add 48ul round 2 mix to each PCR tube.

  2. Add 2 ul of first round product (see section 6.1).

  3. Briefly spin in microcentrifuge (pulse for 5sec).

  4. Transfer tubes to the PCR machine.

  5. Add tubes to thermocycler and cycle using the following programme:

94oC 4min X1

94oC 1min

42oC 2min X30

72oC 1 min
72oC 7min X1
15oC Hold

7.3. VP7 semi- nested consensus PCR (for sequence typing)17
1. Prepare PCR mix for N + 2 (N = No. of tubes in test)

1x mix


10X buffer II (Invitrogen) 4.5ul

50mM MgCl2 2.0ul

dNTPs (10mM) 1.0ul

Taq Polymerase (5U/ul) (Invitrogen) 0.2ul

primer VP7-F18 (20pmoles/ul) 1.0ul

primer VP7-RINT (40pmoles/ul)19 1.0ul

RNase-free H2O 35.3ul

45.0ul


2. Add 45ul of PCR mix to each tube.

3. Add 2ul 1st Round amplicon from (see section 6.1).

4. Briefly spin in microcentrifuge (pulse for 5sec).

5. Transfer tubes to the PCR machine.

6. Add tubes to thermocycler and cycle at the following temperatures for the

following times:



94oC 2min X1

94oC 1min

50oC 1min X35

72oC 1min
72oC 7min X1
15oC Hold
7.4 P-Typing consensus PCR (VP4)
1. Prepare 1st round PCR mix for N + 2 (N = No. of tubes in test)

1x mix


10X buffer II (Invitrogen) 4.5ul

50mM MgCl2 2.5ul

dNTPs (10mM) 1.0ul

Taq Polymerase (5U/ul) (Invitrogen) 0.2ul

Primer VP4F20 (20pmoles/ul) 1.0ul

Primer VP4R (20pmoles/ul) 1.0ul

RNase-free H2O 34.8ul

45.0ul


2. Add 45ul to each tube.

3. Add 5ul cDNA.

4. Briefly spin in microcentrifuge (pulse for 5sec).

5. Transfer tubes to the PCR machine.

6. Add tubes to thermocycler and cycle at the following temperatures for the

following times :


94oC 2min X1

94oC 1min

50oC 1min X35

72oC 1 min


72oC 7min X1
15oC Hold
7.5. P-Typing multiplex PCR
1. Prepare 2nd round mix for N + 2 (N = No. of tubes in test)

1x mix


10X buffer II (Invitrogen) 4.8ul

50mM MgCl2 2.5ul

dNTPs (10mM) 1.0ul

Taq Polymerase (5U/ul) (Invitrogen) 0.2ul

primer VP4F21 (20pmoles/ul) 1.0ul

primer P[4] (20pmoles/ul) 1.0ul

primer P[6] (20pmoles/ul) 1.0ul

primer P[8]22 (20pmoles/ul) 1.0ul

primer P[9] (20pmoles/ul) 1.0ul

primer P[10] (20pmoles/ul) 1.0ul

primer P[11]23 (20pmoles/ul) 1.0ul

RNase-free H2O 32.5ul

48.0ul
2. Add 48ul 2nd round mix to a new 0.2ml tube

3. Add 2ul of first round product (see section 6.4).

4. Briefly spin in microcentrifuge (5sec).

5. Transfer tubes to PCR machine room.

6. Add tubes to thermocycler and cycle at the following temperatures for the following times:
94oC 4min X1

94oC 1min

45oC 2min X30

72oC 1 min


72oC 7min X1
15oC Hold


8. Primer sequences

VP6 oligonucleotide primers (Iturriza Gómara et al 2002, J Virol)

VP6-F 5’ GAC GGV GCR ACT ACA TGG T 3’


VP6-R 5’ GTC CAA TTC ATN CCT GGT G 3’

Product: 382bp


VP6probe for real-time PCR (Taqman) (Iturriza et al unpublished)

VP6probe FAM 5’ CCA CCR AAY ATG ACR CCA GCN GTA 3’ MGB

NSP4 oligonucleotide primers (Ciarlet et al 2000, Virology)

NSP4-F 5’ GGC TTT TAA AAG TTC TGT TCC GAG 3’


NSP4-R 5’ GGT CAC ACT AAG ACC ATT CC 3’

Product size: 743bp

V= A or C or G

Y= C or T

R= A or G

N= A or T or C or G

W=A or T
G-typing oligonucleotide primers


  1. First round consensus primers (Iturriza Gómara et al, 2001, J Clin Microbiol)

VP7-F 5' ATG TAT GGT ATT GAA TAT ACC AC 3' (nt 51-71)

VP7-R 5' AAC TTG CCA CCA TTT TTT CC 3' (nt 914-932)

Product size: 881bp




  1. Second round typing primers: All serotyping primers are included in the mix

[Gouvea et al., 1990, J Clin Microbiol; Iturriza Gómara et al, 2004, J Clin Microbiol; Aladin et al., unpublished]
Genotype G1:

G1 5' CAA GTA CTC AAA TCA ATG ATG G 3' (nt 314-335)

product size; 618bp

Genotype G2:

G2 5' CAA TGA TAT TAA CAC ATT TTC TGT G 3' (nt 411-435)

product size; 521bp

Genotype G3:

G3 5' ACG AAC TCA ACA CGA GAG G 3' (nt 250-269)

product size; 682bp

Genotype G4:

G4 5' CGT TTC TGG TGA GGA GTT G 3' (nt 480-499)

product Size; 452bp

Genotype G8:

G8 5' TTR TCG CAC CAT TTG TGA AAT 3' (nt 176-198)

product size; 756bp

Genotype G9:

G9 5’ CTT GAT GTG ACT AYA AAT AC 3’ (nt 757-776)

product size; 179bp

Genotype G10:

G10 5’ ATG TCA GAC TAC ARA TAC TGG 3’ (nt 666-687)

product size; 266bp

Genotype G12


G12 5¢ GGT TAT GTA ATC CGA TGG ACG 3¢ (nt 548 -567)

product size; 396bp



  1. VP7 nested consensus primer (for sequence typing)

VP7-RINT 5’ ANA YNG ANC CWG TYG GCC A 3’ (nt 331-344)

Product size; 293bp

Y= C or T

W= A or T

R= A or G



P-typing oligonucleotide primers
1. First round consensus primers [Gentsch et al., 1992, J Clin Microbiol] 
VP4F 5' TAT GCT CCA GTN AAT TGG 3' (nt 132-149)

VP4R 5' ATT GCA TTT CTT TCC ATA ATG 3' (nt 775-795)

Product size: 663bp


2. Second round typing primers: All serotyping primers are included in the mix

[Gentsch et al., 1992, J Clin Microbiol;Iturriza-Gómara et al., 2000, J Virol; 2004, J Clin Microbiol]


P[4] (previously genogroup 2)

2T-1 5' CTA TTG TTA GAG GTT AGA GTC 3' (nt 474-494)

product size; 483bp

P[6] (previously genogroup 3)

3T-1 5' TGT TGA TTA GTT GGA TTC AA 3' (nt 259-278)

product size; 267bp

P[8] (previously genogroup 1)

1T-1D 5’ TCT ACT GGR TTR ACN TGC 3’ (nt 339-356)

product size; 345bp

P[9] (previously genogroup 4)

4T-1 5' TGA GAC ATG CAA TTG GAC 3' (nt 385-402)

product size; 391bp

P[10] (previously genogroup 5)

5T-1 5' ATC ATA GTT AGT AGT CGG 3' (nt 575-594)

product size; 583bp

P[11]

P[11] 5' GTA AAC ATC CAG AAT GTG 3' (nt 305-323)

product size; 312bp

N=A or T or G or C

R= A or G

Diagrammatic illustration of VP7 and VP4 specific primer positions and type-specific amplicon sizes.



Group A rotavirus gene 9 (VP7)

1


1062

NA


5'

3'

51

932

176

250

314

411

666

480

757


Amplification products


first round copy of gene 9







881bp

Genotype G8


756bp

Genotype G3


Genotype G1



682bp

618bp


Genotype G2


521bp




primers

Genotype G4


452bp

266bp

Genotype G9

396bp

Genotype G12

Genotype G10



Group A rotavirus gene 4 (VP4)




179bp


132-149

1

2359

775-795

494

402

356

278

NT

594

328


5'

3'

Amplification products




663bp

First Round Amplicon







146bp

P[6]



191bp

P[11]


224bp

P[8]





270bp

P[9]



362bp

P [4]



462bp

P[10]

9 RNA extraction reagents
a. Preparation of solutions
1M Tris
Dissolve 121.1g of Tris base in 800ml of H2O

Adjust the pH to the desired value by adding

concentrated HCl

pH HCl


7.4 ~70ml

7.6 ~60ml

8.0 ~42ml
0.5M EDTA pH 8.0
Add 186.1g of disodium EDTA to 800ml H2O

Adjust the pH to 8.0 with NaOH (~ 20g NaOH

pellets)

L6 Buffer


guanidinium isothiocyanate (GTC) 60g

0.1M Tris-HCl pH 6.4 50ml

0.2M EDTA pH 8.0 11ml

Triton X-100 1.3g


L2 Buffer
GTC 180g

0.1M Tris-HCl pH 6.4 150ml


70% ethanol
Ethanol 70ml

cell culture grade distilled H2O 30ml

b. Preparation of size fractionated silica
1. Add 60g silicon dioxide, SiO2 (Sigma; S-5631) to demineralised water to a total volume of 500ml in a measuring glass cylinder.

2. Allow the silica to sediment under gravity for 24h at room temperature

3. Extract 430ml of supernatant and add demineralised water to 500ml and shake vigorously.

4. Sediment for 24h at room temperature.

5. Extract 440ml supernatant and add 600ul HCl (32%, w/v) to adjust the silica suspension to pH 2.0.

Note: Wear visor, apron and gloves when handling concentrated acids.


6. Aliquot the silica suspension in 4ml volumes in glass bijoux bottles and sterilise by autoclaving.

c. Preparation of RNase-free deionised water


1. Add 100ul of diethylpyrocarbonate (DEPC)(Sigma: D-5758) to 100ml of

cell culture grade deionised water.


NOTE: Perform the addition in a fume cupboard.
2. Incubate for >12hr at 37oC.

3. Autoclave for 15min.


d. Random primers (Invitrogen: 48190011)


Add 450ul of RNase-free water to aliquot,

use 1ul/50ul reaction volume.

e. TE buffer
Tris-HCl, pH 7.4 10mM

EDTA, pH 8.0 1mM


f. Sample buffer (gel loading buffer)
Ficoll 10%

Orange G 0.25%

in TE buffer
g. Ethidium bromide (EtBr)
10mg/ml in TE buffer (if adding to the gel, 20ulEtBr solution in 50ml agarose gel)

10. Disposal of toxic waste

Waste should be discarded in accordance with local guidelines. The following identifies toxic substances used in the procedures described.



Ethidium bromide


1. Ethidium bromide staining solution should be collected and stored for disposal

2. Ethidium bromide stained gels should be discarded in a

labelled discard jar and stored for disposal



Guanidinium isothiocyanate

Add GTC to an equal volume of 10M NaOH and store for disposal


NOTE: GTC will release cyanate gas under acidic conditions

Solvents
Collect ethanol and acetone in a labelled Winchester bottle and store for disposal

11. Agarose- gel Electrophoresis
1. Add 2g of UltraPure™ Agarose 100 (Invotrogne Ct No 16550-100) (or an appropriate gel for the analysis of nucleic acid amplicons of <1Kb) to 100ml 1X TBE for a midi gel to give a 2% gel.

2. Melt in microwave at full power for 2 minutes.


Microwave procedure

Caution: Any microwaved solution may become superheated and boil vigorously when moved or touched. Use extreme care in handling. Remove the boiling solution from the microwave oven, allow to stand for a few seconds at room temperature and release the air by gentle swirling. Heat, using several short, 20 to 30 second intervals with gentle swirling between pulses to resuspend the powder.




  1. Cool to 45oC then pour into gel plate (size 14 cm [W] x 16 cm [L])24

fitted with two 22-28 slot combs (to give a well of 20ul capacity).

  1. Add 10ul of PCR product to 10ul sample buffer in a microtitre plate.

  2. Remove the comb and end pieces and add 20ul of size markers25 or diluted

sample to the appropriate well.

6. Place the gel plate in the gel tank and add 1X TBE level with the gel (do not flood the plate at this stage)

7. Run the products into the gel for 5min at a constant voltage of 150V.

8. Flood the gel with 1X TBE making sure it is fully submerged



9. Electrophorese at constant Voltage (between 5 and 8 V/cm).


1 An animal rotavirus is used as a positive control in order to monitor laboratory contamination.

2 It is important to resuspend the silica completely between wash steps.

3 An animal rotavirus is used as a positive control in order to monitor laboratory contamination.

4 RT and PCR conditions have been optimised using Invitrogen reagents. Re-optimisation may be required if other reagents are used

5 See section 9

6 This PCR is designed for detecting rotavirus RNA in clinical samples, and can be used for confirmation of rotavirus ELISA-equivocal results and SG determination when followed by sequencing.

7 See section 8 for primer sequences

8 See section 8 for primer sequences

9 This PCR is designed for NSP4-genotype characterisation when followed by sequencing.

10 See section 8 for primer sequences

11 These primers are preferred to those of Gouvea et al, as they provide increased sensitivity and sufficient DNA for direct sequencing if the genotype in not determined in the 2nd round multiplex reaction. See section 8 for primer sequences

12 See section 8 for primer sequences

13 The position and sequence of the G3 primer is different from that previously published by Gouvea et al. These changes have been introduced in order to prevent cross-reactivity between G3 and G10 primers and targets.

14 The G9 primer has been modified to take account of the genetic drift of G9 strains from the prototype strain.

15 A G10 primer has been included as increased numbers of G10 strains have been isolated in India in recent years.

16 A G12 primer has been included as increased numbers of G12 strains have been isolated in India in recent years.

15 The G8 and G12 primers have been modified to overcome mistyping

17 This PCR provides increased sensitivity and should be used in conjunction with DNA sequencing for samples which fail to G-type using the method described in section 7.2.

18 See section 8 for primer sequences

19 40 pmoles of primer should be used to take account of its degenerate nature.

20 See section 8 for primer sequences

21 See section 8 for primer sequences

22 The P[8] primer has been modified to take account of the genetic drift of P[8] strains from the prototype strain.

23 A P[11] primer has been included as increased numbers of P[11] strains have been isolated in India in recent years.

24 Avoid the use of mini gels for genotyping 2nd round PCRs as the resolution may not be sufficient to differentiate genotypes.

25 Use a 100 bp ladder for DNA fragments of between 100 and 1000 bp.



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