TABLE. Oligonucleotides designed and used in this study.
| Oligo | Sequence (5’- 3’) | Start | Stop |
| OROV_L_56_FNF | TTGCTCAACCARTATCGRAATAGGAT | 56 | 81 |
| OROV_L_174_FNF | CTGCAAAYCTTGAGTAYAGAAATGATG | 174 | 200 |
| OROV_L_621_FNR | TCAATCCATGGCAATGTCATTGT | 621 | 599 |
| OROV_M_2185_FNF | TCCCAAATCTAATCCTTTTACYGAT | 2185 | 2209 |
| OROV_M_2864_FNF | AGTATAGATGTACAAGGTACAGAATC | 2864 | 2889 |
| OROV_M_3564_FNR | TTCCTTCTCATAGCATGGCAT | 3564 | 3544 |
| OROV_S_6_FNF | TGTACTCCACAATTCAAAACAT | 6 | 27 |
| OROV_S_133_FNF | ACGGACAAGTGCTCAATGCT | 133 | 152 |
| OROV_S_728_FNR | TCCGAATTGGCGCAAGAAGT | 728 | 709 |
| Assay | Primer pairs | Size (bp) | |
| L - 1st PCR (55ºC) | OROV_L_56_FNF + OROV_L_621_FNR | 566 | |
| L - semi-nested (50ºC) | OROV_L_174_FNF + OROV_L_621_FNR | 448 | |
| M - 1st PCR (55ºC) | OROV_M_2185_FNF + OROV_M_3564_FNR | 1380 | |
| M - semi-nested (55ºC) | OROV_M_2864_FNF + OROV_M_3564_FNR | 701 | |
| S - 1st PCR (58ºC) | OROV_S_6_FNF + OROV_S_728_FNR | 723 | |
| S - semi-nested (58ºC) | OROV_S_133_FNF + OROV_S_728_FNR | 596 | |
Start/Stop positions refers to the nucleotide position of OROV GenBank reference sequence NC_005776.1 (segment L), NC_005775.1 (segment M), and NC_005777.1 (segment S). To increase sensitivity, we developed semi-nested reactions for each genome segment. All 1st polymerase chain reaction (PCR) reactions followed the same program: 94ºC for 2 min for enzyme activation; 35 cycles (94ºC for 30 s, 55 or 58ºC for 30 s, and 72ºC during 1 min/Kb), a final step at 72ºC for 5 min. For semi-nested reactions: 94ºC for 2 min for enzyme activation; 30 cycles (94ºC for 30 s, 50, 55 or 58ºC for 30 s, and 72ºC for 1 min), a final step at 72ºC for 5 min. All primers used in this study were synthesised by IDT DNA Technology, USA.