Table 1.
Sequences of primers and probes used in this study.
| Assay | Primer/Probe | Sequence (5′–3′) | Target | Amplicon | Reference |
|---|---|---|---|---|---|
| Singleplex SADS-CoV PCR | |||||
| Assay 1 | SADS-N-F1 | CTGACTGTTGTTGAGGTTAC | N | 155 bp | Zhou L et al., J Virol Methods. 2018, 255:66–70 [35] |
| SADS-N-R1 | TCTGCCAAAGCTTGTTTAAC | N | |||
| SADS-N-Prb1 | FAM/TCACAGTCT/ZEN/CGTTCTCGCAATCA/3IABkFQ | N | |||
| Assay 2 | SADS-N-F2 | GCACTTTTATTACCTTGGTA | N | 144 bp | Xu et al., Transbound Emerg Dis. 2019, 66:119–130 [36] |
| SADS-N-R2 | GTAGCAGGTTCTTTGTTAC | N | |||
| SADS-N-Prb2 | FAM/TCCTCACGC/ZEN/AGATGCTCCTT/3IABkFQ | N | |||
| Assay 3 | SADS-N-F3 | CCAGGCCTCAAAGTGGTAAAAA | N | 85 bp | This study |
| SADS-N-R3 | TGCTTACGAGCCGGTTTAGG | N | |||
| SADS-N-Prb3 | FAM/ACCCAAACC/ZEN/AAGAAGCAGAGCTGTCTCAC/3IABkFQ | N | |||
| Assay 4 | SADS-N-F4 | TTGGCAGACTTGGGCATAGC | N | 85 bp | This study |
| SADS-N-R4 | GTGAGACAGCTCTGCTTCTTGGT | N | |||
| SADS-N-Prb4 | FAM/TCCAGGCCT/ZEN/CAAAGTGTAAAAATACACCC/3IABkFQ | N | |||
| Singleplex PDCoV PCR | |||||
| Assay 1 | PDCoV-M-RTF | CGACCACATGGCTCCAATTC | M | 70 bp | Chen et al., Virology. 2015, 482:51–59 [30] |
| PDCoV-M-RTR | CAGCTCTTGCCCATGTAGCTT | M | |||
| PDCoV-M-RTP | FAM/CACACCAGT/ZEN/CGTTAAGCATGGCAAGC/3IABkFQ | M | |||
| Assay 2 | PDCoV-N-RTF | CCTACTACTGACGCGTCTTGGTT | N | 75 bp | Chen et al., Emerg Microbes Infect. 2018, 7:105 [31] |
| PDCoV-N-RTR | TGCCACGAAACTGAGGATGA | N | |||
| PDCoV-N-RTP | VIC/TGCTCAAAGCTCAAAAC/MGB | N | |||
| Assay 3 | PDCoV-N-F2 | CCAGACATGTGCCTGGTGTT | N | 68 bp | This study |
| PDCoV-N-R2 | CCCYGCCTGAAAGTTGCT | N | |||
| PDCoV-N-Prb2 | ABY/ARATGCTTTTCGCTGGCCACCTTG/QSY | N | |||
| Singleplex TGEV PCR | |||||
| Assay 1 | TGEV-N-F | TGCCATGAACAAACCAAC | N | 81 bp | Huang et al., Appl Microbiol Biotechnol. 2019, 103:4943–4952 [42] |
| TGEV-N-R | GGCACTTTACCATCGAAT | N | |||
| TGEV-N-Prb | VIC/TAGCACCACGACTACCAAGC/MGB-3′ | N | |||
| Assay 2 | TGEV-S-F2 | GTGGTAATATGYTRTATGGCYTACAA | S | 101 bp | This study |
| TGEV-S-R2 | GCCAGACCATTGATTTTCAAAACT | S | |||
| TGEV-S-Prb2 | VIC/TTGCTTATTTACATGGTGCYAGT/MGB | S | |||
| Singleplex PEDV PCR | |||||
| Assay 1 | PEDV-N306-F | CGCAAAGACTGAACCCACTAACCT | N | 198 bp | Madson et al., Vet Microbiol. 2014, 174:60–68 [28] |
| PEDV-N503-R | TTGCCTCTGTTGTTACTTGGAGAT | N | |||
| PEDV-N469-Prb | FAM/TGTTGCCATTACCACGACTCCTGC/QSY | N | |||
| Assay 2 | PEDV-N1195-F | GAAGAGGCCATCTACGATGATGT | N | 75 bp | This study |
| PEDV-N1269-R | AACAGCTGTGTCCCATTCCAA | N | |||
| PEDV-N1221-Prb | JUN/TGTGCCATCTGATGTGACTCATGCCA/QSY | N | |||
| PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex PCR | |||||
| PEDV-N1195-F | GAAGAGGCCATCTACGATGATGT | N | 75 bp | This study | |
| PEDV-N1269-R | AACAGCTGTGTCCCATTCCAA | N | |||
| PEDV-N1221-Prb | JUN/TGTGCCATCTGATGTGACTCATGCCA/QSY | N | |||
| PDCoV-N-F2 | CCAGACATGTGCCTGGTGTT | N | 68 bp | This study | |
| PDCoV-N-R2 | CCCYGCCTGAAAGTTGCT | N | |||
| PDCoV-N-Prb2 | ABY/ARATGCTTTTCGCTGGCCACCTTG/QSY | N | |||
| TGEV-S-F2 | GTGGTAATATGYTRTATGGCYTACAA | S | 101 bp | This study | |
| TGEV-S-R2 | GCCAGACCATTGATTTTCAAAACT | S | |||
| TGEV-S-Prb2 | VIC/TTGCTTATTTACATGGTGCYAGT/MGB | S | |||
| SADS-N-F3 | CCAGGCCTCAAAGTGGTAAAAA | N | 85 bp | This study | |
| SADS-N-R3 | TGCTTACGAGCCGGTTTAGG | N | |||
| SADS-N-Prb3 | FAM/ACCCAAACC/ZEN/AAGAAGCAGAGCTGTCTCAC/QSY | N | |||
Note: The singleplex PCR assays used to develop the 5-plex PCR are shown as highlighted.