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

Using nasopharyngeal swabs, saliva, urine, and rectal swabs from patients enrolled in our COVID-19 research protocol at the Yale-New Haven Hospital, we found that more samples had lower Ct values (more efficient virus detection) using the 2019-nCoV_N1 primer-probe set as compared to 2019-nCoV_N2 ( Fig. 4A ). Interestingly, with the N1 set, samples with a Ct value of approximately 40 and samples that were not detected (ND) were clearly discrete groups, whereas several samples that were not detected by the N1 set were in the 41-43 Ct range for the N2 set ( Fig. 5B ). This further supports our observation that the US CDC 2019-nCoV_N2 primer-probe set produces some non-specific amplification. However, when we look at the US CDC assay outcomes, which take into account both the N1 and N2 results, only 1 out of 172 tests was deemed inconclusive due to N1 being negative (>40 Ct) and N2 being positive (<40 Ct) due to non-specific amplification ( Fig. 5C ). When a more stringent Ct value cut-off of 38 was implemented, we did not detect any inconclusive results where N2 was the only positive set. In fact, we found more inconclusive results where N1 was the only positive set at both 40 Ct (3/172) and 38 Ct (5/172) cut-offs ( Fig. 5C ), likely because the 2019-nCoV_N1 primer-probe set is more sensitive ( Figs. 4, 5A, 5B ). Overall, we generated inconclusive results from less than 3% of the tested clinical samples using the US CDC primer-probe sets, indicating that background cross-reactivity does not have a large impact on the testing outcomes.