Analytical sensitivity and efficiency comparisons of SARS-COV-2 qRT-PCR assays

Chantal B.F. Vogels; Anderson F. Brito; Anne Louise Wyllie; Joseph R Fauver; Isabel M. Ott; Chaney C. Kalinich; Mary E. Petrone; Marie-Louise Landry; Ellen F. Foxman; Nathan D. Grubaugh

We generated RNA transcript standards for each of the five genes targeted by the diagnostic qRT-PCR assays using T7 transcription. A detailed protocol can be found here 10 . Briefly, cDNA was synthesized from full-length SARS-CoV-2 RNA (WA1_USA strain from UTMB; GenBank: MN985325). Using PCR, we amplified the nsp10, RdRp, nsp14, E, and N genes with specifically designed primers ( Supplemental Table 1 ). We purified PCR products using the Mag-Bind TotalPure NGS kit (Omega Bio-tek, Norcross, GA, USA) and quantified products using the Qubit High Sensitivity DNA kit (ThermoFisher Scientific, Waltham, MA, USA). We determined fragment sizes using the DNA 1000 kit on the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). After quantification, we transcribed 100-200 ng of each purified PCR product into RNA using the Megascript T7 kit (ThermoFisher Scientific). We quantified RNA transcripts using the Qubit High sensitivity RNA kit (ThermoFisher Scientific) and checked quality using the Bioanalyzer RNA pico 6000 kit. For each of the RNA transcript standards ( Supplemental Table 2 ), we calculated the number of genome copies per µL using Avogadro's number. We generated a genomic annotation plot with all newly generated RNA transcript standards and the nine tested primer-probe sets based on the NC_045512 reference genome using the DNA Features Viewer Python package ( Fig. 1A ) 16 . We generated standard curves for each combination of primer-probe set with its corresponding RNA transcript standard ( Fig. 1B ) , using standardized qRT-PCR conditions as described below.