Sars-Cov-2
Introduction
As the Covid-19 pandemic hit Norway in the march 2020 the health authorities dedicated funds to increase research on the topic. Since the testing strategy for Sars-Cov-2 virus had been established by cDNA synthesis from virus RNA and detection by Taqman PCR the need for chemistry was evident. Furthermore, a supply shortage from commercial sources was observed. Thus, if in-house enzyme, reverse transcriptase and Taq polymerase could be produced and tested, that could potentially be beneficial.
Primers and probes published from different labs was extracted from the World Health Organization (WHO) webpage and used for testing of in-house chemistry. Several assays that detect the 2019-nCoV have been development. Some assays may detect only the novel virus and some may also detect other strains (e.g. SARS-CoV) that are genetically similar. Common for the assays were the detection method by rtPCR with "Syber Green" or with TaqMan probe.
Molecular assays to diagnose 2019-nCoV
| Links and primer "name" | Annealing | Primer set # |
"Forward" "Reverse" "Probe" |
Reporter dye | 5' - 3' | Quencher |
| Source | ||||||
| ORF1ab | ||||||
| ORF1ab（F） | 1 | F | CCCTGTGGGTTTTACACTTAA | |||
| ORF1ab（R） | 1 | R | ACGATTGTGCATCAGCTGA | |||
| ORF1ab（P） | 1 | P | FAM- | CCGTCTGCGGTATGTGGAAAGGTTATGG | BHQ1 | |
| N-gene | ||||||
| N（F） | 2 | F | GGGGAACTTCTCCTGCTAGAAT | |||
| N（R） | 2 | R | CAGACATTTTGCTCTCAAGCTG | |||
| N（P） | 2 | P | FAM- | TTGCTGCTGCTTGACAGATT | TAMRA | |
| Source | ||||||
| RdRP_SARSr-F2 | 58°C | 3 | F | GTGARATGGTCATGTGTGGCGG | ||
| RdRP_SARSr-R1 | 3 | R | CARATGTTAAASACACTATTAGCATA | |||
| RdRP_SARSr-P2 | 3 | P | FAM- | CAGGTGGAACCTCATCAGGAGATGC | BBQ | |
| RdRP_SARSr-P1 | 3 | P | FAM- | CCAGGTGGWACRTCATCMGGTGATGC | BBQ | |
| E_Sarbeco_F1 | 4 | F | ACAGGTACGTTAATAGTTAATAGCGT | |||
| E_Sarbeco_R2 | 4 | R | ATATTGCAGCAGTACGCACACA | |||
| E_Sarbeco_P1 | 4 | P | FAM- | ACACTAGCCATCCTTACTGCGCTTCG | BBQ | |
| Source | ||||||
| Assay 1 (Target: ORF1b-nsp14) | ||||||
| (HKU-ORF1b-nsp14F): | 5 | F | TGGGGYTTTACRGGTAACCT | |||
| (HKU-ORF1b-nsp14R): | 5 | R | AACRCGCTTAACAAAGCACTC | |||
| (HKU-ORF1b-nsp141P): | 5 | P | FAM- | TAGTTGTGATGCWATCATGACTAG | TAMRA | |
| Assay 2 (Target: N) | ||||||
| (HKU-NF) | 6 | F | TAATCAGACAAGGAACTGATTA | |||
| (HKU-NR) | 6 | R | CGAAGGTGTGACTTCCATG | |||
| Probe (HKU-NP): | 6 | P | FAM- | GCAAATTGTGCAATTTGCGG | TAMRA | |
| Source | ||||||
| WH-NIC N-F | 7 | F | CGTTTGGTGGACCCTCAGAT | |||
| WH-NIC N-R | 7 | R | CCCCACTGCGTTCTCCATT | |||
| WH-NIC N-P | 7 | P | FAM- | CAACTGGCAGTAACCA | BHQ1 | |
| Source | ||||||
| ORF1a set | ||||||
| NIID_WH-1_F501 | 60°C | 8 | F | TTCGGATGCTCGAACTGCACC | ||
| NIID_WH-1_R913 | 8 | R | CTTTACCAGCACGTGCTAGAAGG | |||
| NIID_WH-1_F509 | 60°C | 9 | F | CTCGAACTGCACCTCATGG | ||
| NIID_WH-1_R854 | 9 | R | CAGAAGTTGTTATCGACATAGC | |||
| NIID_WH-1_Seq_F519 | 60°C | 10 | F | ACCTCATGGTCATGTTATGG | ||
| NIID_WH-1_Seq_R840 | 10 | R | GACATAGCGAGTGTATGCC | |||
| S set | ||||||
| WuhanCoV-spk1-f | 60°C | 11 | F | TTGGCAAAATTCAAGACTCACTTT | ||
| WuhanCoV-spk2-r | 11 | R | TGTGGTTCATAAAAATTCCTTTGTG | |||
| NIID_WH-1_F24381 | 60°C | 12 | F | TCAAGACTCACTTTCTTCCAC | ||
| NIID_WH-1_R24873 | 12 | R | ATTTGAAACAAAGACACCTTCAC | |||
| NIID_WH-1_Seq_F24383 | 60°C | 13 | F | AAGACTCACTTTCTTCCACAG | ||
| NIID_WH-1_Seq_R24865 | 13 | R | CAAAGACACCTTCACGAGG | |||
| NIID_2019-nCOV_N_F2 | 60°C | 14 | F | AAATTTTGGGGACCAGGAAC | ||
| NIID_2019-nCOV_N_R2 | 14 | R | TGGCAGCTGTGTAGGTCAAC | |||
| NIID_2019-nCOV_N_P2 | 14 | P | FAM- | ATGTCGCGCATTGGCATGGA | ||
| Source | ||||||
| 2019-nCoV_N1-F | 55°C | 15 | F | GACCCCAAAATCAGCGAAAT | ||
| 2019-nCoV_N1-R | 15 | R | TCTGGTTACTGCCAGTTGAATCTG | |||
| 2019-nCoV_N1-P | 15 | P | FAM- | ACCCCGCATTACGTTTGGTGGACC | BHQ1 | |
| 2019-nCoV_N2-F | 55°C | 16 | F | TTACAAACATTGGCCGCAAA | ||
| 2019-nCoV_N2-R | 16 | R | GCGCGACATTCCGAAGAA | |||
| 2019-nCoV_N2-P | 16 | P | FAM- | ACAATTTGCCCCCAGCGCTTCAG | BHQ1 | |
| 2019-nCoV_N3-F | 55°C | 17 | F | GGGAGCCTTGAATACACCAAAA | ||
| 2019-nCoV_N3-R | 17 | R | TGTAGCACGATTGCAGCATTG | |||
| 2019-nCoV_N3-P | 17 | P | FAM- | AYCACATTGGCACCCGCAATCCTG | BHQ1 | |
| RNAse-RP-F | 55°C | 18 | F | AGATTTGGACCTGCGAGCG | ||
| RNAse-RP-R | 18 | R | GAGCGGCTGTCTCCACAAGT | |||
| RNAse-RP-P | 18 | P | FAM- | TTCTGACCTGAAGGCTCTGCGCG | BHQ1 | |
| Source | ||||||
| RdRp gene/nCov_IP2 | ||||||
| nCoV_IP2-12669Fw | 58°C | 19 | F | ATGAGCTTAGTCCTGTTG | ||
| nCoV_IP2-12759Rv | 19 | R | CTCCCTTTGTTGTGTTGT | |||
| nCoV_IP2-12696bProbe(+) | 19 | P | HEX- | AGATGTCTTGTGCTGCCGGTA | BHQ1 | |
| RdRp | ||||||
| nCoV_IP4-14059Fw | 58°C | 20 | F | GGTAACTGGTATGATTTCG | ||
| nCoV_IP4-14146Rv | 20 | R | CTGGTCAAGGTTAATATAGG | |||
| nCoV_IP4-14084Probe(+) | 20 | P | FAM- | TCATACAAACCACGCCAGG | BHQ1 | |
| E_gene / E_Sarbeco | ||||||
| E_Sarbeco_F1 | 58°C | 21 | F | ACAGGTACGTTAATAGTTAATAGCGT | ||
| E_Sarbeco_R2 | 21 | R | ATATTGCAGCAGTACGCACACA | |||
| E_Sarbeco_P1 | 21 | P | FAM- | ACACTAGCCATCCTTACTGCGCTTCG | BHQ1 |
Location of primers
The location of the primers and probes are mapped with https://www.bioinformatics.org/sms2/primer_map.html and can be viewed in this PDF. Some of the primers in the table above does not match the NCBI Reference Sequence: NC_045512.2
Link description