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

To further investigate the relatively low performance of the RdRp-SARSr (Charité) primer-probe set, we compared our standardized primer-probe concentrations with the recommended concentrations in the confirmatory (Probe 1 and Probe 2) and discriminatory (Probe 2 only) RdRp-SARSr (Charité) assays. We deviated from the recommended concentrations in the original assays to make a fair comparison across primer-probe sets, using 500 nM of each primer and 250 nM of probe 2. To investigate the effect of primer-probe concentration on the ability to detect SARS-CoV-2, we made a direct comparison between ( 1 ) our standardized primer (500 nM) and probe (250 nM) concentrations, ( 2 ) the recommended concentrations of 600 nM of forward primer, 800 nM of reverse primer, and 100 nM of probe 1 and 2 (confirmatory assay), and ( 3 ) the recommended concentrations of 600 nM of forward primer, 800 nM of reverse primer, and 200 nM of probe 2 (discriminatory assay) per reaction 5 . We found that adjusting the primer-probe concentrations or using the combination of probes 1 and 2 did not increase SARS-CoV-2 RNA detection when using 10-fold serial dilutions of our RdRp RNA transcripts, or full-length SARS-CoV-2 RNA from cell culture ( Fig. 5 ). The Charité Institute of Virology Universitätsmedizin Berlin assay is designed to use the E-Sarbeco primer-probes as an initial screening assay, and the RdRp-SARSr primer-probes as a confirmatory test 5 .