Table 1.
Primers & probes for the developed multiplex allele-specific qRT-PCR assays.
| Assay | Allele | Forward primer (5’-3’) | Probe (5’-3’) | Reverse primer (5’-3’) | Amplicon (bp) | |
|---|---|---|---|---|---|---|
| Prototype allele targeting reactions | Assay 1 | NSP1: S135R | TAATAAAGGAGCTGGTGGCCATTGT | FAM-CGGCGCCGATCTAAAGTCATTTGACTTAGGCGAC-BHQ1 | CCACAGAAGTTGTTATCGACATAGCG | 194 |
| NSP6: I189V | CTGTCATGTTTTTGGCCAGAGCTA | VIC-TTGGCCTCTTTTGTTTACTCAACCGCTACTTTAGACTGACTC-BHQ1 | GTGGGAGTAGTCCCTGTGAATTC | 242 | ||
| Spike: A27S | CCAGAACTCAATTACCCCGTG | Cy3-CTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGT-BHQ2 | CCATCATTAAATGGTAGGACAGGGTTATC | 207 | ||
| Spike: S371L | CTGTGTTGCTGATTATTCTGTCCTATATAAATC | Cy5-AGGTGATGAAGTCAGACAAATCGCTCCAGGGC-BHQ2 | GTAAATTGCGGACATACTTATCGGC | 179 | ||
| Assay 2 | NSP6: F108L | GGATATGGTTGATACTAGTTTGTCTGGATT | FAM-CCTTATGACAGCAAGAACTGTGTATGATGATGGTGCTAGGA BHQ1 | GGAAATGGCTTGATCTAAAGCATTACC | 195 | |
| Spike: G446S | CTTGGAATTCTAACAATCTTGATTCTAAGGATG | VIC-CAACTGAAATCTATCAGGCCGGTAGCACACCTTGT BHQ1 | CATTAGTGGGTTGGAAACCATATGATTGT | 197 | ||
| Spike: T547K | ACAAATGTGTCAATTTCAACTTCAATGGTTTTAC | Cy3-CTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACAC-BHQ2 | ATGGTGTAATGTCAAGAATCTCAAGTGTC | 161 | ||
| Spike: L981F | TTGGTGCAATTTCAAGTGTTTTAAATGATAACC | Cy5-CTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCA-BHQ2 | TTGATTGTCCAAGTACACACTCTGAC | 203 | ||
| Assay 3 | NSP3: T24I | GTGCAAGGTTACAAGAGTGTGAATATGAC | FAM-TGAGAAGTGCTCTGCCTATACAGTTGAACTCGGTACAGAAG BHQ1 | GGTGTAAGTAATTCAGATACTGGTTGCAAAG | 178 | |
| NSP3: L1266I | GTAGGAGACATTATACTTAAACCAGCAAATAATACTT | VIC-CAGAAGAGGTTGGCCACACAGATCTAATGGCTGC BHQ1 | GGGTTTTCAAACCTAATACTCTAGATAATTCATTAGG | 150 | ||
| Spike: V213G | AATATATTCTAAGCACACGCCTATTAATTTTGT | Cy3-CGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACC-BHQ2 | CACCAGGAGTCAAATAACTTCTATGTAAAGC | 151 | ||
| Spike: R408S | GATTCATTTGTAATTAGAGGTGATGAAGTCTGA | Cy5-CGCTCCAGGGCAAACTGGAAAGATTGCTG-BHQ2 | GGTAATTATAATTACCACCAACCTTAGAATCAAG | 162 | ||
| Mutated allele targeting reactions | Assay 1 | NSP1: S135R | TAATAAAGGAGCTGGTGGCCATTGG | FAM-CGGCGCCGATCTAAAGTCATTTGACTTAGGCGAC BHQ1 | CCACAGAAGTTGTTATCGACATAGCG | 194 |
| NSP6: I189V | TGTCATGTTTTTGGCCAGAGCTG | VIC-TTGGCCTCTTTTGTTTACTCAACCGCTACTTTAGACTGACTC BHQ1 | GTGGGAGTAGTCCCTGTGAATTC | 241 | ||
| Spike: A27S | AACCAGAACTCAATTACCCCGTT | Cy3-CTTGGTTCCATGCTATACATGTCTCTGGGACCAATGGT-BHQ2 | CCATCATTAAATGGTAGGACAGGGTTATC | 209 | ||
| Spike: S371L | CTGTGTTGCTGATTATTCTGTCCTATATAAACT | Cy5-AGGTGATGAAGTCAGACAAATCGCTCCAGGGC-BHQ2 | GTAAATTGCGGACATACTTATCGGC | 179 | ||
| Assay 2 | NSP6: F108L | GGATATGGTTGATACTAGTTTGTCTGGATA | FAM-CCTTATGACAGCAAGAACTGTGTATGATGATGGTGCTAGGA BHQ1 | GGAAATGGCTTGATCTAAAGCATTACC | 195 | |
| Spike: G446S | CTTGGAATTCTAACAATCTTGATTCTAAGGATA | VIC-CAACTGAAATCTATCAGGCCGGTAGCACACCTTGT BHQ1 | CATTAGTGGGTTGGAAACCATATGATTGT | 197 | ||
| Spike: T547K | ACAAATGTGTCAATTTCAACTTCAATGGTTTTAA | Cy3-CTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACAC-BHQ2 | ATGGTGTAATGTCAAGAATCTCAAGTGTC | 161 | ||
| Spike: L981F | TTGGTGCAATTTCAAGTGTTTTAAATGATAACT | Cy5-CTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCA-BHQ2 | GTTGAGTCACATATGTCTGCAAACTTTG | 203 | ||
| Assay 3 | NSP3: T24I | GTGCAAGGTTACAAGAGTGTGAATATGAT | FAM-TGAGAAGTGCTCTGCCTATACAGTTGAACTCGGTACAGAAG BHQ1 | GGTGTAAGTAATTCAGATACTGGTTGCAAAG | 178 | |
| NSP3: L1266I | GTAGGAGACATTATACTTAAACCAGCAAATAATACTA | VIC-CAGAAGAGGTTGGCCACACAGATCTAATGGCTGC BHQ1 | GGGTTTTCAAACCTAATACTCTAGATAATTCATTAGG | 150 | ||
| Spike: V213G | AATATATTCTAAGCACACGCCTATTAATTTTGG | Cy3-CGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACC-BHQ2 | CACCAGGAGTCAAATAACTTCTATGTAAAGC | 151 | ||
| Spike: R408S | GATTCATTTGTAATTAGAGGTGATGAAGTCTGC | Cy5-CGCTCCAGGGCAAACTGGAAAGATTGCTG-BHQ2 | GGTAATTATAATTACCACCAACCTTAGAATCAAG | 162 | ||