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Protocol
Swine vesicular disease virus PriProET two-step real-time PCR
Keywords: swine vesicular disease virus, SVD, SVDV, PriProET, RT-PCR
This SOP describes a two-step protocol for PriProET real-time PCR amplification of swine vesicular disease viruses (SVDV). A key point of the assay is tolerance toward mutations in the probe region.
This SOP describes a two-step protocol for PriProET real-time PCR amplification of swine vesicular disease viruses (SVDV). The assay amplifies the 3D-gene of any of SVDV strain while heterologous virus strains including Coxsackievirus B5 remain negative. The sensitivity of assay is five copies of viral genome equivalents. A key point of the assay is tolerance toward mutations in the probe region. Melting curve analysis directly after PCR, with determination of probe melting point, confirms specific hybridisation of the SVDV strains.
The protocol was optimised using ABI 7700 instrument (Applied Biosystems, USA), but later was adopted for RotorGene (Corbett Research, Australia). Both instruments works well with the system, but RotorGene, like many other modern real-time PCR machines, has friendly user format and simplifies analysis of results.
Required Materials
- pd(N)6 primer , for cDNA synthesis
- dNTP kit (2 mM) , for cDNA synthesis
- 5 x First strand buffer , for cDNA synthesis
- RNAguard™ Ribonuclease Inhibitor (Porcine) , for cDNA synthesis
- M-MLV Reverse Transcriptase , for cDNA synthesis
Instructions
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RNA extraction: (L2BHWT)
Manual TRIzol extraction according to the manufacturer’s instructions was used in the current protocol, but any isolation method that gives good RNA yield could be used instead.
This report describes a simple and reliable extraction technique using commercial reagents (TRIzol) to provide RNA for subsequent reverse transcription and specific PCR amplification. Samples suitable for testing include field samples and cells suspected to contain RNA viruses of vesicular disease. Extraction of total RNA from such samples is necessary to facilitate the processes of reverse transcription and polymerase chain reaction (RT-PCR) amplification.
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Risk assessment
Health and Safety and Other Risks:
Disposable gloves and a laboratory coat should be worn to avoid contact with skin and clothes when using TRIzol Reagent as it contains phenol. Tubes containing waste TRIzol Reagent must be placed into a suitable container for appropriate disposal.
RNA extraction must be carried out in a location separate from areas where reverse transcription and PCR amplification are carried out to avoid contamination of stock reagents.
Disposable gloves must be worn throughout the technique as RNases from the skin have an adverse affect on RNA. Precipitated extracted RNA pellets may be very small or invisible; care must be taken to avoid accidental discard. Extracted RNA should be stored at -90 to -50oC in a monitored freezer.
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Responsibilities
Health and Safety:
This protocol should be used in accordance with the current Health and Safety Policy and other relevant instructions as issued by the Institute for Animal Health. Due consideration must be given to National standards and regulations.
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Procedure
- Ensure any samples frozen and previously stored in TRIzol are thawed. Incubate for 5 minutes at approximately 22°C (room temperature).
- Label a fresh 2 ml skirted Sarstedt tube corresponding to each test sample to be processed; add 200 ?l of chloroform to each 2 ml tube followed by 1.0 ml of the TRIzol solution containing the sample.
- Vortex mix each tube for 10-15 seconds.
- Centrifuge the tubes for 15 minutes at 13,000 rpm at 2-8°C.
- Label a fresh 1.5 ml conical tube for each test sample and add 1.0 ?l of glycogen to each.
- Remove 500 ?l of the top phase (clear phase) from the centrifuged tubes (from stage 5.4) and add to the corresponding glycogen-containing tubes. Add 500 ?l of isopropyl alcohol to each tube.
- Vortex mix each tube for a few seconds.
- Incubate the tubes at approximately 4°C (on ice) for 10 minutes.
- Orientate the tubes in the centrifuge using the tube label so that the RNA pellet will be in an expected position. Centrifuge the tubes for 10 minutes at 13,000 rpm at 2-8°C.
- Discard the supernatant from the tubes and then add 1.0 ml of 70% ethanol to each one.
- Vortex mix the tubes for a few seconds.
- Orientate the tubes in the centrifuge as in stage 5.9 and centrifuge for 10 minutes at 13,000 rpm at 2-8°C.
- Remove as much supernatant as possible using gentle vacuum suction (or pipetting) without disturbing the RNA pellet. Air dry each tube for 2-3 minutes at approximately 22°C. Resolubilise each RNA pellet by adding 20 ?l of nuclease-free water to each tube and gentle mixing.
- Maintain the RNA samples on ice and immediately proceed with RT-PCR or store them at -90°C to -50°C until required.
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Results
Results will only become known after analysis of the subsequent reverse transcription and PCR amplification procedures.
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Maintenance
If pipettes subsequently fail calibration checks but final results are satisfactory according to the controls, the Line Manager or Operator may still deem the results acceptable
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cDNA synthesis
cDNA was produced in 25 ?l reaction volume using 5 ?l RNA, 1 ?l random hexamers pd(N)6, 0.02 U, (Amersham, Uppsala, Sweden), 2.5 ?l dNTPs (2 mM), 5 ?l 5X First Strand Buffer, 1 ?l RNAguard (1000 U, Amersham Bioscience, USA), and 1 ?l M-MLV reverse transcriptase (200 U, Ambion, Austin, Texas, USA). The reaction was incubated for 5 min at 22°C, followed by 90 min at 37°C, and the enzyme was finally inactivated by heating for 5 min at 95°C.
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Real-time PCR (L2B9FW)
This SOP describes a two-step protocol for PriProET real-time PCR amplification of swine vesicular disease viruses (SVDV). The assay amplifies the 3D-gene of any of SVDV strain while heterologous virus strains including Coxsackievirus B5 remain negative. The sensitivity of assay is five copies of viral genome equivalents. A key point of the assay is tolerance toward mutations in the probe region. Melting curve analysis directly after PCR, with determination of probe melting point, confirms specific hybridisation of the SVDV strains.
The protocol was optimised using ABI 7700 instrument (Applied Biosystems, USA), but later was adopted for RotorGene (Corbett Research, Australia). Both instruments works well with the system, but RotorGene, like many other modern real-time PCR machines, has friendly user format and simplifies analysis of results.
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RT-PCR
General aspects:
The amplification is based on the TITANIUM Taq DNA polymerase kit (Clontech) with a total reaction volume of 25 µl.
Controls: PC (positive control), NTC (no template control)
After production of the master mix, 23 µl portions are aliquoted into each reaction tube and 2 µl of template is added.
Master mix preparation:
The volume of the master mix depends on the number of extracted samples plus the number of controls (PC; NTC) plus one-two extra reactions.
Before dividing up the master mix into portions of 23 µl per tube, the master mix must be mixed thoroughly and pulse spanned for 5 sec.
Addition of template cDNA:
2 µl template cDNA is added to each tube with 23 µl master mix and close the reaction tubes.
Spin down reaction mix (optional).
Cycler program:
Preliminary denaturation 95°C 2 min Denaturation 95°C 15 sec Annealing 60°C 15 sec 55 cycles Elongation 72°C 15 sec Denaturation 95°C 15 sec Probe melting profile
96 cycles of [50°C for 10 sec with auto-increments of 0.5°C] for ABI 7700 ?or 40°C->95°C for RotorGene.
Fluorescence channel is FAM for ABI 7700. Filter set for RotorGene is 470nm/610hp (emission/detection)
Fluorescence data are collected in the annealing phase and during the probe melting profile
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Analysis of data
A Ct value of approx. 25 should be detectable for PC and a specific probe melting point of 57,5 -72,5°C (depends on the number of mutations in the probe region). Each mutation in the probe region decreases melting point on 5°C.
No Ct value or melting point should be detectable for NTC.
The analysis of field samples is possible if all controls respond as expected.
A suspected SVDV sample is considered as positive in the real-time PCR, if a Ct value is detected for the sample and/or if the FAM fluorescence increases significantly over the base level.
For confirmation of a positive SVDV specific real-time PCR result the probe melting profile has to display a specific probe melting point of 70°C±2.5°C corresponding to a perfect probe match, while Tm =65±2.5°C and Tm =60±2.5°C corresponding to one or two mutations, respectively.
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References
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Development of a real-time PCR assay based on primer-probe energy transfer for the detection of swine vesicular disease virus
,Author: M. Hakhverdyan, T. B. Rasmussen, P. Thorén, Å. Uttenthal, S. Belák | Date: December 2006