Table 1. Design of two PCR assays for detection of influenza A(H5Nx) clade 2.3.4.4b .
| Code | Oligonucleotide | Length (bp) | GC (%) | Tm (°C) | Amplicon size |
|---|---|---|---|---|---|
| JRC-HA | |||||
| HA01-Fwd | 5’-ACTGGGCTCAGAAATAGTCCTC-3’ | 22 | 50.0 | 59.2 | 139 bp |
| HA01-Rev | 5’-TCCCCTGCTCATTGCTATGATG-3’ | 22 | 50.0 | 60.2 | |
| HA01-Prb | 5’-FAM-AGAGGGAGGATGGCAGGGAATGGTT-QSY7–3’ | 25 | 56.0 | 60.9 | |
| Amplicon | ACTGGGCTCAGAAATAGTCCTCTAAGAGAAAAGAGAAGAAAAAGAGGTCTGTTTGGGGCGATAGCAGGGTTTATAGAGGGAGGATGGCAGGGAATGGTTGATGGTTGGTATGGGTACCATCATAGCAATGAGCAGGGGA | ||||
| JRC-MP | |||||
| MP01-Fwd | 5’-TGTCTTTGCAGGGAAGAACACC-3’ | 22 | 50.0 | 61.0 | 114 bp |
| MP01-Rev | 5’-ACGGTGAGCGTGAACACAAATC-3’ | 22 | 50.0 | 61.9 | |
| MP01-Prb | 5’-VIC-ATCTTGAGGCTCTCATGGAATGGCT-QSY7–3’ | 25 | 48.0 | 57.8 | |
| Amplicon | TGTCTTTGCAGGGAAGAACACCGATCTTGAGGCTCTCATGGAATGGCTAAAGACAAGACCAATCCTGTCACCTCTGACTAAGGGGATTTTGGGATTTGTGTTCACGCTCACCGT | ||||
Fwd: forward primer; JRC: Joint Research Centre; Prb: probe; Rev: reverse primer; Tm: melting temperature.
For each assay, the reported amplicon is the most frequently observed sequence targeted by the assay in the United States outbreak sequence segments deposited in GISAID (December 2024).