Oligo name
Sequence (5′–3′)
Working concentration (μM)
Primer Hofm_BTV_IVI_F2
TGG AYA AAG CRA TGT CAA A
20
Primer Hofm_BTV_IVI_R2
ACR TCA TCA CGA AAC GCT TC
20
Probe Hofm_BTV_IVI_Pa
FAM-ARG CTG CAT TCG CAT CGT ACG C-Tamra
5
8.
Aerosol-resistant pipette tips.
9.
Adhesive PCR film.
10.
Marker pens (waterproof) and adhesive labels.
2.2 Equipment
1.
PCR cabinet.
2.
Calibrated pipettes.
3.
Freezer (−5 to −30 °C).
4.
Refrigerator (1–8 °C).
5.
Ice bucket with ice.
6.
Large capacity centrifuge.
7.
Real-time PCR machine.
3 Methods
3.1 Sample Preparation
1.
dsRNA from BTV test samples should be prepared as per standard methods (e.g., Qiagen/Roche viral nucleic acid extraction kits) and stored at −50 to −90 °C. BTV negative and positive control samples of known origin should also be prepared (see Note 2 ).
3.2 Assay Setup
1.
In a PCR clean room, prepare the one-step real-time RT-PCR reaction master mix according to Table 2. Sufficient volume of the master mix should be prepared to allow for testing of the required number of samples (see Note 3 ).
Table 2
Composition of the master mix for one-step real-time RT-PCR
|
Master mix composition |
Volume for 1 reaction (μl) |
|---|---|
|
2× Superscript III RT-PCR reaction mix |
12.5 |
|
Primer Hofm_BTV_IVI_F2 (20 μM)a |
0.5 |
|
Primer Hofm_BTV_IVI_R2 (20 μM)a |
0.5 |
|
Probe Hofm_BTV_IVI_P (5 μM)a |
1.0 |
|
RNase-free water |
2.5 |
|
2.5 μM ROX l |
0.5 |
|
50 mM MgSO4 |
1.0 |
|
Superscript III RT/Platinum Taq |
0.5 |
|
Total volume |
19 |
2.
Working in a clean laminar flow cabinet (see Note 4 ), add all reagents to a suitable container (e.g., microcentrifuge tube) allowing for the total volume of reagents (see Note 5 ). Mix the reagents gently with a pipette. Once outside the PCR clean room, maintain the master mix on ice and shielded from light.
3.
In a class II safety cabinet, carefully aliquot 6 μl of extracted RNA for both samples and controls into an optical reaction PCR plate following a planned layout (see Note 6 ). Alternatively, use PCR strips/tubes that are suitable for a real-time PCR machine.
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