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

The most potentially problematic mismatch is in the RdRp-SARSr reverse primer ( Fig. 6B ), which likely explains our sensitivity issues with this set ( Figs. 2, 3, 6 ) . Oddly, the mismatch is not derived from a new variant that has arisen, but rather that the primer contains a degenerate nucleotide (S, binds with G or C) at position 12, and 990 of the 992 SARS-CoV-2 genomes encode for a T at this genome position ( Fig. 6B ). This degenerate nucleotide appears to have been added to help the primer anneal to SARS-CoV and bat-SARS-related CoV genomes 5 , seemingly to the detriment of consistent SARS-CoV-2 detection. Earlier in the outbreak, before hundreds of SARS-CoV-2 genomes became available, non-SARS-CoV-2 data were used to infer genetic diversity that could be anticipated during the outbreak. As a result, several of the primers contain degenerate nucleotides ( Supplemental Table 3 ). For RdRp-SARSr, adjusting the primer (S→A) may resolve its low sensitivity.