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Diagnostic detection of Wuhan coronavirus 2019 by real-time RTPCR

Table 1. Primers and probes  Optimized concentrations are mol per liter of final reaction mix.  (e.g., 1.5 microliters of a 10 micromolar (uM) primer stock solution per 25 microliter (ul) total reaction volume yields a final concentration of 600 nanomol per liter (nM) as indicated in the table) -note that standard, non-optimized reaction conditions as indicated by suppliers of one-step RT-PCR kits will generally yield sufficient sensitivity-   Assay/ Use Oligonucleotide ID Sequence (5’–3’) Comment
RdRP gene
RdRP_SARSr-F2 GTGARATGGTCATGTGTGGCGG use 600 nM per reaction
RdRP_SARSr-R1 CARATGTTAAASACACTATTAGCATA use 800 nM per reaction
RdRP_SARSr-P2 FAM-CAGGTGGAACCTCATCAGGAGATGC- BBQ
Specific for Wuhan-CoV, will not detect SARSCoV use 100 nM per reaction and mix with P1
RdRP_SARSr-P1 FAMCCAGGTGGWACRTCATCMGGTGATGC- BBQ
Pan Sarbeco-Probe, will detect Wuhan virus, SARS-CoV and bat-SARS-related CoVs use 100 nM per reaction and mix with P2
E gene E_Sarbeco_F1 ACAGGTACGTTAATAGTTAATAGCGT use 400 nM per reaction
E_Sarbeco_R2 ATATTGCAGCAGTACGCACACA use 400 nM per reaction
E_Sarbeco_P1 FAM-ACACTAGCCATCCTTACTGCGCTTCG- BBQ
use 200 nM per reaction
N gene N_Sarbeco_F1 CACATTGGCACCCGCAATC use 600 nM per reaction
N_Sarbeco_R1 GAGGAACGAGAAGAGGCTTG use 800 nM per reaction
N_Sarbeco_P1 FAM-ACTTCCTCAAGGAACAACATTGCCA- BBQ
use 200 nM per reaction
W is A/T; R is G/A; M is A/C ; FAM, 6-carboxyfluorescein; BBQ, blackberry quencher


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