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

Of the variants that we detected in the primer-probe regions, we only found four in more than 30 of the 992 SARS-CoV-2 genomes (>3%, Fig. 6B ). Most notable was a stretch of three nucleotide substitutions (GGG→AAC) at genome positions 28,881-28,883, which occur in the three first positions of the CCDC-N forward primer binding site. While these substitutions define a large clade that includes~13% of the available SARS-CoV-2 genomes and has been detected in numerous countries 14 , their position on the 5' location of the primer may not be detrimental to sequence annealing and amplification. The other high frequency variant that we detected was T→C substitution at the 8 th position of the binding region of the 2019-nCoV_N3 forward primer, a substitution found in 39 genomes (position 28,688). While this primer could be problematic for detecting viruses with this variant, the 2019-nCoV_N3 set has already been removed from the US CDC assay. We found another seven variants in only five or fewer genomes (<0.5%, Fig. 6B ), and their minor frequency at present does not pose a major concern for viral detection. This scenario may change if those variants increase in frequency: most of them lie in the second half of the primer binding region, and may decrease primer sensitivity 15 . The WA1_USA strain (GenBank: MN985325) that we used for our comparisons did not contain any of these variants.