Table 4. Primers and probes evaluated during the study.
| Author | Target (nt) | Oligonucleotides | Sequence (5’-3’)* | Tm (°C) |
|---|---|---|---|---|
| Pre-existing in-house |
NS3 (103) | Forward | 6399-TYG-AYG-CRA-GRG-TTT-ATG-CAG | 61–68 |
| Probe 1 | Fam-AGT-GGT-TTA-AGG-ATT-TTG-CAG-C-Tamra | 62 | ||
| Probe 2 | Fam-AGT-GGT-TCA-ARG-ACT-TTG-CAG-C-Tamra | 64–66 | ||
| Reverse | 6502-CAT-RCG-ACC-GAG-CAC-CTC-TA | 64–67 | ||
| Pyke et al 2004 |
NS5 (62) | Forward | 10230-ATC-TGG-TGY-GGY-AGT-CTC-A | 61–67 |
| Probe | Fam-CGG-AAC-GCG-ATC-CAG-GGC-AA-Tamra | 69 | ||
| Reverse | 10292-CGC-GTA-GAT-GTT-CTC-AGC-CC | 65 | ||
| Yang et al 2004 |
3' UTR (146) | Forward | 10764-GGT-GTA-AGG-ACT-AGA-GGT-TAG-AGG | 64 |
| Probe | Fam-CCC-GTG-GAA-ACA-ACA-TCA-TGC-GGC-Tamra | 70 | ||
| Reverse | 10910-ATT-CCC-AGG-TGT-CAA-TAT-GCT-GTT | 66 | ||
| Shirato et al 2005 | 5' UTR (75) | Forward | 80-AGA-ACG-GAA-GAY-AAC-CAT-GAC-TAA-A | 64–66 |
| Probe | Fam-ACC-AGG-AGG-GCC-CGG-MGB NFQ | 81 | ||
| Reverse | 155-CCG-CGT-TTC-AGC-ATA-TTG-AT | 62 | ||
| New (this study) | ||||
| NS2A (all Genotypes)–v1 | NS2A (116) | Forward | 3563-AGC-TGG-GCC-TTC-TGG-T | 64 |
| Probe | Fam-CTT-CGC-AAG-AGG-TGG-ACG-GCC-A-Tamra | 70 | ||
| Reverse | 3675-CCC-AAG-CAT-CAG-CAC-AAG | 62 | ||
| NS2A)–v2 | NS2A (112) | Forward | 3563-AGC-TGG-GCC-TTC-TGG-T | 64 |
| Probe | Fam- TGG-CCG-TCC-ACC-TCT-TGC-GAA-G -Tamra | 70 | ||
| Reverse | 3675-CCC-AAG-CAT-CAG-CAC-AAG | 62 | ||
| NS5 (G 1)–v1 | NS5 (315) | Forward | 9925-GDG-CTG-GAT-GGA-ATG-TGA | 61–63 |
| Probe | Fam-AGG-AGA-GTG-GAT-GAC-CAC-MGB NFQ | 75 | ||
| Reverse | 10240-CCA-CAC-CAG-ATG-TCC-TC | 60 | ||
| NS5 (G 1)–v2 | NS5 (315) | Forward | 9925-GDG-CTG-GAT-GGA-ATG-TGA | 61–63 |
| Probe | Fam-AGG-AGA-GTG-GAT-GAC-YAC-MGB NFQ | 72–76 | ||
| Reverse | 10240-CCA-CAC-CAG-ATG-TCC-TC | 60 | ||
| NS3 (G 3)–v1 | NS3 (141) | Forward | 5726-GCA-ATG-TGC-CTC-CAA-AGA-GC | 65 |
| Probe | Fam-TCC-TAT-GAY-ACA-GAA-TAY-CCA-AA-MGB NFQ | 73–78 | ||
| Reverse | 5884-GTC-GAT-GAC-CCT-GCT-CGC | 66 | ||
| NS3 (G 3)–v2 | NS3 (158) | Forward | 5726-GCA-ATG-TGY-CTC-CAA-AGA-GC | 63–66 |
| Probe | Fam-TCC-TAT-GAY-ACA-GAA-TAY-CCA-AA-MGB NFQ | 73–78 | ||
| Reverse | 5884-GTC-GAT-GAC-CCT-GCT-CGC | 66 |
*Position of the oligonucleotides is related to the Nakayama strain, Accession number EF571853.
Tm: melting temperature calculated using IDTNA tool https://www.idtdna.com/calc/analyzer setting parameters as follows: Target type RNA, Oligo Conc 0.4μM, Na Conc 50mM, Mg Conc 3mM, dNTPs Conc 0.8mM. Tms for MGB (minor groove binder) probes include an additional 15°C.
A more extensive list of all oligonucleotides evaluated in-silico is included in Table C in S2 File.
NS2Av2 was designed as NS2Av1 with a modification of the probe sequence as its reverse complement.
NS5v2 was designed as NS5v1 with a modification of the probe sequence, with a degeneracy Y inserted instead of C at position 16.
NS3v2 was designed as NS3v1 with a modification of the forward primer sequence, with a degeneracy Y inserted instead of C at position 9.
NS = gene coding for non-structural protein. G = genotype