Table 2.
Details of oligonucleotide sequences, fluorophores, optimal concentrations and annealing temperatures used for the detection of SARS-CoV-2 targeting Nsp10, N-gene, Nsp12, JUN and EICAS, collectively referred to as CoV2-ID.
| Target | Target | Amplicon (bp) | Oligonucleotides (5′-3′) | Final conc. (µM) | Ta (°C) |
|---|---|---|---|---|---|
| SARS-CoV-2 | Nsp10 | 85 | F: GGATCAAGAATCCTTTGGTGG | 1 | 64.0 |
| R: GTCACAAAATCCTTTAGGATTTGGA | 1 | ||||
| Pr: FAM-CATCGTGTTGTCTGTACTGCCGTTGCC-Q | 0.4 | ||||
| N-gene | 88 | F: GCTGCTAGACAGATTGAAC | 1 | 60.5 | |
| R: AGCAGATTTCTTAGTGACAGTTTG | 1 | ||||
| Pr1:-TR-ATGTCTGGTAAAGGCCAACAACAACA-Q | 0.4 | ||||
| Pr2: TR/FAM-TCTGGTAAAGGCCAACAACAACAAGG-Q | 0.4 | ||||
| Nsp12 | 96 | F: CATCCCTACTATAACTCAAATGAA | 1 | 62.0 | |
| R: GTCATAGTACTACAGATAGAGACAC | 1 | ||||
| Pr: FAM-TGCAAAGAATAGAGCTCGCACCGT-Q | 0.4 | ||||
| Mu-Nsp10 | 85 | (10Mu) GGATCAAGAATCTTTTGGTGG | 1 | 61.0 | |
| Mu-N-gene | 88 | (NA) GCTGCTTGACAGTTTGAAC | 1 | 62.0 | |
| (NB) GCTGCTTGATAGATTGAAC | 1 | 60.3 | |||
| MuN: TR-TTTGGTAAAGGCCAACAACAACAAGG-Q | 0.4 | ||||
| D614G mutation | 104 | F: CACCAGGAACAAATACTTC | 1 | ||
| R: CCAAGTAGGAGTAAGTTGA | 1 | ||||
| WT: FAM-ctttAtcAggAtgTtaact-Q | 0.4 | ||||
| Mu: HEX-ctttAtcAggGtgTtaact-Q | 0.4 | ||||
| mRNA control | JUN | 88 | F: CGCCTGATAATCCAGTCCA | 1 | 60.5 |
| R: GCTCATCTGTCACGTTCTTG | 1 | ||||
| Pr:- Cy5-CACATCACCACCACGCCGACC-Q | 0.4 | ||||
| EICAS1 | – | 58 | F: AACAACCACACCAAAAC | ||
| R: GGAGGTTTTAGTTTGG | |||||
| Pr: HEX-CACACAACACCAACAAAACCAAACA-Q | |||||
| rArArCrArArCrCrArCrArCrCrArArArArCrCrArCrArCrArArCrArCrCrArArCrArArArArCrCrArArArCrArCrCrArArArCrUrArArArArCrCrUrCrC | |||||
| EICAS2 | – | 65 | Pr: HEX-CACACAACACCAACAAAACCAAACA-Q | ||
| rCrGrCrCrUrGrArUrArArUrCrCrArGrUrCrCrArUrCrArCrArCrArArCrArCrCrArArCrArArArArCrCrArArArCrArCrArArGrArArCrGrUrGrArCrArGrArUrGrArGrC | |||||
All oligonucleotides are listed in the 5′-3′ direction. Nucleotides that differ from the SARS-CoV-2 reference sequence, but have been detected at low frequency are shown in red. Oligonucleotides are based on the following accession numbers: Reference SARS-CoV-2: NC_045512; mutant SARS-CoV-2: MT412262 (F-primer 10Mu); MT607612/481905/496997/467255/467251 (F-primers NA and NB); MT506889/506904/506907 (Probe MuN); c-JUN: NM_002228.4.