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Protocol
Bluetongue SYBR-Green RT-PCR
Keywords: bloetounge virus, BTV, RT-PCR, CYBR Green
Describes detection of most of BTV serotypes reported in the Mediterranean basin by one-step RT-PCR.
The protocol described here allows the detection of most of BTV serotypes reported in the Mediterranean basin by one-step RT-PCR (Reverse Transcriptase Polymerase Chain Reaction). So far, a 77 bp within the NS1 segment belonging to serotypes 1, 2, 4, 8 and 9 have been successfully amplified. The amplification of BTV16 with the primers described here is still ongoing. The set of primers used in this protocol has been designed by Partner 4, UCM, Madrid.
Required Materials
- Brilliant® II SYBR® Green QRT-PCR Master Mix Kit, 1-Step
- Milli-Q water , or any other PCR grade water.
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Primers
- BTVNS1-F (20 ?M) , forward primer
- BTVNS1-R (20 ?M) , reverse primer
Instructions
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RNA extraction (L2BHWT)
RNA isolation is performed with Trizol Reagent, following the manufacturer’s instructions.
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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Real-time PCR
General aspects:
The amplification is based on the SYBR® Green QRT-PCR Master Mix One-Step kit (Stratagene) with a total reaction volume of 25µl.
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 two extra reactions.
Before dividing up the master mix into portions of 23 µl per tube, the master mix must be mixed thoroughly.
Addition of template RNA:
2µl template RNA is added to each tube with 23µl master mix.
After addition of template RNA, close the reaction tubes and spin down the solution.
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Cycler program
Reverse Transcription 48°C 30 min Inactivation RT /Activation Taq 95°C 2 min 40 cycles Denaturation 95°C 30 sec Annealing 56°C 30 sec Elongation 72°C 30 sec Denaturation 95°C 5 min Probe melting profile 60°C->95°C
Fluorescence channels are ROX (negative control) and SYBR GREEN.
Fluorescence data are collected in the annealing phase and during the probe melting profile
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Analysis of generated fluorescence data
General aspects:
ROX channel can be used for normalisation of the SYBR Green fluorescence
SYBR Green channel:
- A Ct value of approx. 20 should be detectable for PC and a specific probe melting point of 78°C should be observed.
- 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 BTV sample is considered positive in the real-time RT-PCR, if a Ct value is detected for the sample and/or if the SYBR Green fluorescence increases significantly over the base level.
- For confirmation of a positive BTV specific real-time PCR result, the probe melting profile has to display a specific probe melting point of 75-78°C.
