Primers and Probes

Primers and probes were selected from highly conserved regions of the SARS-CoV-2 genome based on ≈4,000 sequences available in GISAID (https://www.gisaid.org) in March 2020 (Table 1). Primer Express 3.0.1 software (Thermo Fisher Scientific, https://www.thermofisher.com) was used to design primers that had annealing temperatures of ≈60°C and probes that had annealing temperatures of ≈68°C.

The multiplex assay probes were synthesized by using ZEN or TAO Double-Quenched Probes labeled at the 5′ end using reporter 6-carboxyfluorescein (FAM) for InfA, Yakima Yellow for InfB, Texas Red-XN for SARS-CoV-2, and Cyanine 5 (Cy 5) for RP targets (Integrated DNA Technologies, Inc., https://www.idtdna.com). The InfA and InfB probes were quenched with ZEN between nucleotides 9 and 10 and with Iowa Black FQ at the 3′ end; the SARS-CoV-2 and RP probes were quenched with TAO between nucleotides 9 and 10 and with Iowa Black RQ at the 3′ end (Integrated DNA Technologies, Inc.). Primers and Taqman hydrolysis probes were synthesized by Integrated DNA Technologies and the CDC Biotechnology Core Facility Branch (Division of Scientific Resources, National Center for Emerging and Zoonotic Infectious Diseases; Atlanta, Georgia, USA).