Table 1.
The primers and probes used for the multiplex asymmetric RT-PCR assay in this study.
| Primer | Sequence (5′–3′) | Location | Tm (°C) | Probe | Sequence (5′–3′) | Location | Reference strain GenBank Number |
|---|---|---|---|---|---|---|---|
| M-forward | TCTTCTAACCGAGGTCGAAACG | 31–52 | 62.7 | M-CP | TTGTATTCACGCACACCGTGC - C6 S-S | 210–230 | HM142730.1 |
| M- reverse | CGTCTACGCTGCAGTCCT | 241–258 | 57.9 | M-SP | Fc - CCCAGTGAGCGAGG | 230–243 | |
| H1- forward | TAATAGCACCATGGTATGCTTT | 785–806 | 54.7 | H1-CP | TCATTGAAGGGGGATGGACAGGA - C6 S-S | 1049–1071 | FN436023.1 |
| H1-SP | Fc - ATGATAGATGGGTGGTATGG | 1072–1091 | |||||
| H2- forward | ATAGAAGGRGGATGGCAAGG | 1057–1076 | 56.0 | H2-CP | GCAGCAGACAAAGAGTCCA- C6 S-S | 1166–1184 | KP767619.1 |
| H2-SP | Fc - CTCAAAAGGCAATTGATGGAATCACCAAC | 1185–1213 | |||||
| H3- forward | TTCACTTGGACAGGAGTAAC | 421–440 | 51.9 | H3-CP | ATGCCAAACAATGACAATTTTGAC - C6 S-S | 550–573 | KY415635.1 |
| H3-SP | Fc - AAACTATACATCTGGGG | 574–590 | |||||
| H5- forward | ATCATTGACAAAATGAACACTCAR | 1201–1225 | 57.6 | H5-CP | ATGGARGACGGATTCCTAGATGTCTGG - C6 S-S | 1288–1314 | DQ095621.2 |
| H5-SP | Fc - ACTTATAAYGCTGAACT | 1315–1331 | |||||
| H7- forward | TAAGCAGCGGCTACAAAG | 1550–1567 | 56.3 | H7-CP | TGGTTTAGCTTCGGGGCATCAT - C6 S-S | 1579–1600 | KP765952.1 |
| H7-SP | Fc - GTTTTCTTCTTCTAGCC | 1601–1617 | |||||
| H9- forward | TCAACAAACTCCACAGAAAC | 79–98 | 49.6 | H9-CP | CTAACAGAAAACAATGTYCCT - C6 S-S | 109–129 | KT699056.1 |
| H9-SP | Fc - GTGACACATGCCAAAGAA | 130–147 | |||||
| H10- forward | ATAGCACCGAGCCGAGTTAG | 803–822 | 60.9 | H10-CP | CACCAATAGACAATAATTGTGAGTCCA - C6 S-S | 861–887 | KY402019.1 |
| H10-SP | Fc - AATGTTTTTGGAGAGGG | 888–904 | |||||
| N1- forward | AGTGCTCCTGTTATCCTGATG | 773–793 | 51.7 | N1-CP | TATATATGCAGTGGAGTTTTCGGAGA - C6 S-S | 886–911 | KF667692.1 |
| N1-SP | Fc - CAATCCACGCCCCAATG | 912–928 | |||||
| N2- forward | AAGGACAACTCAATTAGGCTYTC | 314–336 | 56.2 | N2-CP | ACCAAACAAGTGTGCATAGCAT - C6 S-S | 521–542 | KP767935.1 |
| N2-SP | Fc - GGTCTAGTTCAAGCTG | 543–558 | |||||
| N6- forward | TGCATAGGATGGTCAAGCA | 526–544 | 49.0 | N6-CP | AGGATGTCAATATGCATATCAGGA - C6 S-S | 568–591 | KM251485.1 |
| N6-SP | Fc - CCGAATAACAATGCATC | 592–608 | |||||
| N8- forward | CAATGAAACAGTAAGGGTTGAGA | 159–181 | 55.0 | N8-CP | ATTGTGTGATGCTAAGGGTTTCG - C6 S-S | 264–286 | KY402021.1 |
| N8-SP | Fc - CACCCTTTTCCAAAGAC | 287–303 | |||||
| N9- forward | ACAACCAACACAAGCCAAAC | 148–167 | 57.3 | N9-CP | TTTCAATAACTTAACTAAAGGGCTC - C6 S-S | 237–261 | KP416240.1 |
| N9-SP | Fc - TGTACTATAAATTCATGGC | 262–280 | |||||
| Universal reverse 01 | ATATCGTCTCGTATTAAGTAGAAACAAG | 1716–1743 | 60.6 | / | / | / | FN436023.1 |
| Universal reverse 02 | ATATGGTCTCGTATTAGTAGAAACAAGGAG | 1357–1386 | 61.6 | / | / | / | KF667692.1 |
| RNaseP - forward | AGATTTGGACCTGCGAGCG | / | 55.0 | RNaseP - probe | FAM - TTCTGACCTGAAGGCTCTGCGCG - BHQ-1 | / | / |
| RNaseP - reverse | GAGCGGCTGTCTCCACAAGT | 59.0 | |||||
| 18S rRNA forward | AGCGAAAGCATTTGCCAAGA | / | 53.0 | / | / | / | / |
| 18S rRNA reverse | AGTCTCGTTCGTTATCGGAATT | 57.0 |
CP, capture probe; SP, signal probe; and Fc, ferrocene.