Table 1.
Real-time RT-PCR primers and probes designed and used in this study
| Primer and probe sets | Sequences | Gene target | Location (bp) | GenBank accession no. |
|---|---|---|---|---|
| Designed and used in this study | ||||
| Flu A forward | GGARTGGCTAAAGACAAGACCAATC | Matrix protein | 129–153 | |
| Flu A reverse | GGCATTYTGGACAAAGCGTCTAC | Matrix protein | 227–249 | KC885959.1 |
| Flu A probe | ROX-AGTCCTCGCTCACTGGGCACGGT-BHQ2 | Matrix protein | 199–221 | |
| (H1N1) 2009 forward | GGAAAGAAATGCTGGATCTGGTA | Hemagglutinin | 822–844 | |
| (H1N1) 2009 reverse | ATGGGAGGCTGGTGTTTATAGC | Hemagglutinin | 904–925 | KP019929.1 |
| (H1N1) 2009 probe | TAMRA-TGCAATACAACTTGTCARACACCCGAAGG-BHQ2 | Hemagglutinin | 874–902 | |
| H3 forward | GMAGCAAAGCCTACAGCAACT | Hemagglutinin | 269–289 | |
| H3 reverse | GCCGGATGAGGCAACTAGTG | Hemagglutinin | 329–348 | KP662684.1 |
| H3 probe | HEX-CTTATGATGTGCCGGATTATGCCTCCC-BHQ1 | Hemagglutinin | 296–322 | |
| H7 forward | GAGGCRATGCAAAATAGAATACAGAT | Hemagglutinin | 1510–1535 | |
| H7 reverse | CCGAAGCTAAACCARAGTATCACA | Hemagglutinin | 1569–1592 | KC885956.1 |
| H7 probe | FAM-ACCCRGTCAAACTAAGCAGCGGYTAYAA-BHQ1 | Hemagglutinin | 1538–1565 | |
| RP forward | AGATTTGGACCTGCGAGCG | Ribonuclease P | 50–68 | |
| RP reverse | GAGCGGCTGTCTCCACAAGT | Ribonuclease P | 71–93 | NM_006413.4 |
| RP probe | CY5-TTCTGACCTGAAGGCTCTGCGCG-BHQ2 | Ribonuclease P | 95–114 | |
| Designed in previous study and used in this study | ||||
| Flu A forward | GACCRATCCTGTCACCTCTGAC | Matrix protein | ||
| Flu A reverse | AGGGCATTYTGGACAAAKCGTCTA | Matrix protein | WHO (2013)(17) | |
| Flu A probe | FAM-TGCAGTCCTCGCTCACTGGGCACG-BHQ1 | Matrix protein | ||
| H1 (2009) forward | TGAGATATTCCCCAAGACAAGTTC | Hemagglutinin | ||
| H1 (2009) reverse | TTTGTAGAAGCTTTTTGCTCCAG | Hemagglutinin | Reference (2) | |
| H1 (2009) probe | FAM-TCATGACTCGAACAAAGGTGTAACGG-BHQ1 | Hemagglutinin | ||
| H3 forward | ACCCTCAGTGTGATGGCTTCCAAA | Hemagglutinin | ||
| H3 reverse | TAAGGGAGGCATAATCCGGCACAT | Hemagglutinin | Reference (2) | |
| H3 probe | CY5-ACGCAGCAAAGCCTACAGCAACTGT-BHQ2 | Hemagglutinin | ||
| H7 forward | AGAAATGAAATGGCTCCTGTCAA | Hemagglutinin | ||
| H7 reverse | GGTTTTTTCTTGTATTTTTATATGACTTAG | Hemagglutinin | WHO (2013)(17) | |
| H7 probe | FAM-AGATAATGCTGCATTCCCGCAGATG-BHQ1 | Hemagglutinin | ||
| RP forward | AGATTTGGACCTGCGAGCG | Ribonuclease P | ||
| RP reverse | GAGCGGCTGTCTCCACAAGT | Ribonuclease P | WHO (2013)(17) | |
| RP probe | FAM-TTCTGACCTGAAGGCTCTGCGCG-BHQ1- | Ribonuclease P | ||
Flu A, universal influenza A virus; H1 (2009), pandemic [H1N1] 2009 virus; H3, seasonal H3N2 virus; H7, reassortant avian H7N9 virus; RP, Rnase P gene. HEX is hexachloro-fluorescein phosphoramidite; BHQ is Black Hole Quencher; ROX is carboxy-X-rhodamine; FAM is a proprietary fluorophore similar to 6-carboxyfluorescein fluorophore; CY5 is blue reporter dye xyanocyline; TAMRA is Carboxytetramethylrhodamine dye. The probe consisted of oligonucleotides with the 5′ reporter dye and the 3′ quencher dye