Analytical Validation of a COVID-19 qRT-PCR Detection Assay Using a 384-well Format and Three Extraction Methods

Andrew C Nelson; Benjamin Auch; Matthew Schomaker; Daryl M Gohl; Patrick Grady; Darrell Johnson; Robyn Kincaid; Kylene E Karnuth; Jerry Daniel; Jessica K Fiege; Elizabeth J Fay; Tyler Bold; Ryan A Langlois; Kenneth B Beckman; Sophia Yohe

Our preliminary experiments also included parallel assessment of a 20 µL assay format mimicking the volume setup of the CDC-specified assay. We set up PCR replicates for a subset of samples on both volume formats to compare average Cts and coefficients of variation (CV) between the volume formats. The key objective was to determine if the decreased sample input (2.5 µL) of our 384-well format assay significantly compromised assay performance in comparison to the larger volume (5 µL sample input) format. We observed small shifts in raw Ct values of <0.1 to 0.5 for the N1 and N2 targets (Table 5) . We transformed replicate Ct values to relative quantity (copy number) to calculate the true quantitative CV across data points for each sample/target on the two assay formats ( Table 6 ). The copy number CVs for clinical samples ranged from 6-14%; the synthetic extraction standard CVs (at 200 cp/µL) ranged from 6-21%.