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 study does have several limitations to consider. First, we standardized PCR conditions to make a fair comparison between primer-probes sets used in four common qRT-PCR assays for detection of SARS-CoV-2. By standardizing the concentration of primers and probes, PCR kits, and thermocycler conditions, we deviated from the conditions as recommended by each assay which may have influenced our findings. For instance, we selected an annealing temperature of 55°C which was lower than recommended for the assays developed by Charité (58°C) 5 and HKU (60°C) 4 , but similar to the assay developed by US CDC (55°C) 6 . No specific PCR conditions were reported for the assay developed by the China CDC 7 . The two assays (Charité and HKU) with higher annealing temperatures did not yield background amplification, which suggests that our standardized annealing temperature likely did not have a large effect on our findings. Second, when determining the sensitivity of primer-probe sets, we performed eight replicates at low concentrations of SARS-CoV-2 RNA spiked into (pre-COVID-19) clinical samples. While we evaluated the US CDC using 172 clinical samples collected during the COVID-19 pandemic, more replicates for the other assays are required to accurately determine the lower detection limit. Importantly, sensitivity as reported in our study may not be applicable to other PCR kits or thermocyclers; analytical sensitivities and positive-negative cut-off values should be locally validated when establishing these assays.