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

Our comparative results of primer-probe sets used in qRT-PCR assays indicate some variation in the analytical sensitivities for SARS-CoV-2 detection and ability to differentiate between true negatives and positives at low RNA concentrations. We found that the most sensitive primer-probe sets are E-Sarbeco (Charité), HKU-ORF1 (HKU), HKU-N (HKU), and 2019-nCoV_N1 (US CDC), which could partially (63-75%) detect SARS-CoV-2 at 10 virus copies per μL of RNA. In contrast, the RdRp-SARSr (Charité) primer-probe set had the lowest sensitivity, as also shown by an independent study 13 , likely stemming from a mismatch in the reverse primer. Furthermore, we found background cross-reactivity when testing nasopharyngeal swabs prior to the COVID-19 pandemic using the China CDC and US CDC assays. While the China CDC "N" and "ORF1" primer-probe sets require further investigation, we found that the background cross-reactivity did not affect the outcomes of the US CDC assay when testing clinical samples from the COVID-19 pandemic. Overall, our findings indicate that the qRT-PCR assay from HKU, the screening assay (E-Sarbeco) from Charité, and the updated assay from the US CDC are most reliable for accurate detection of SARS-CoV-2.