Table 3. Composition of reaction mixes for one-step RT-PCR reaction with one set of primers and probe (3a) and two sets of primers and probes (3b).
| Table 3a—One-step RT-PCR reaction mix with one set of primers and probe | |
| Reagent | Amount (μl) |
| Forward primer (10 pm/μl-1) | 1 |
| Reverse primer (10 pm/μl-1) | 1 |
| Probe (5 pm/μl-1) | 1 |
| MgSO4 (50mM) | 1 |
| ROX (1/10 dilution) | 0.5 |
| 1Superscript® III RT/Platinum® Taq Mix 2x reaction mix | 12.5 |
| Nuclease free water (μl) | 5 |
| dsRNA (μl) | 3 |
| Total volume (μl) | 25 |
| Table 3b2- One-step RT-PCR reaction mix with two sets of primers and probes | |
| Forward primer 1 (10 pm/μl-1) | 1 |
| Reverse primer 1 (10 pm/μl-1) | 1 |
| Probe 1 (5 pm/μl-1) | 0.5 |
| Forward primer 2 (10 pm/μl-1) | 1 |
| Reverse primer 2 (10 pm/μl-1) | 1 |
| Probe 2 (5 pm/μl-1) | 0.5 |
| MgSO4 (50mM) | 1 |
| ROX (1/10 dilution) | 0.5 |
| Superscript® III RT/Platinum® Taq Mix 2x reaction mix | 12.5 |
| Nuclease free water (μl) | 3 |
| dsRNA (μl) | 3 |
| Total volume (μl) | 25 |
1SuperScript III/ Platinum Taq One-Step qRT-PCR Kit (Invitrogen, UK).
2The duplex format of the assay is for use with sets of primers and probes for the same serotype (eastern and western strains).