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

A barrier to implementing and validating qRT-PCR molecular assays for SARS-CoV-2 detection was the availability of virus RNA standards. As the full length SARS-CoV-2 RNA is considered as a biological safety level 2 hazard in the US, we generated small RNA transcripts (704-1363 nt) from the non-structural protein 10 (nsp10), RNA-dependent RNA polymerase (RdRp), non-structural protein 14 (nsp14), envelope (E), and nucleocapsid (N) genes spanning each of the primer and probe sets in the China CDC 7 , US CDC 6 , Charité 5 , and HKU 4 assays ( Fig. 1A ; Table 1; Supplemental Tables 1-2 ) 10 . By measuring PCR amplification using 10-fold serial dilutions of our RNA transcript standards, we found the efficiencies of each of the nine primer-probe sets to be above 90% ( Fig. 1B ) , which match the criteria for an efficient qRT-PCR assay 11 . Our RNA transcripts can thus be used for assay validation, positive controls, and standards to quantify viral loads: critical steps for a diagnostic assay. Our protocol to generate the RNA transcripts is openly available 10 , and any clinical or research diagnostic lab can directly request them for free through our lab website ( www.grubaughlab.com ). ( A ) We reverse-transcribed RNA transcript standards for the non-structural protein 10 (nsp10), RNA-dependent RNA polymerase (RdRp), non-structural protein 14 (nsp14), envelope (E), and nucleocapsid (N) genes to be used for validation of nine primer-probe sets used in SARS-CoV-2 qRT-PCR assays. ( B ) We generated standard curves for nine primer-probe sets with 10-fold dilutions . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.