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

Preliminary Assay Characterization An initial pilot experiment aimed to prove acceptable performance of the 384 well assay format with a 10 µL assay volume. We utilized a subset of available clinical biospecimens, synthetic positive extraction controls, and nucleic acid controls to test initial parameters, confirm reproducibility, and determine a initial limit of detection. One aliquot each of four clinical samples (two positive, two negative) and three separate aliquots of a synthetic positive control with 200 viral copies per microliter (EDx_200) were extracted with Qiagen viral RNA reagents (one of the CDC-approved extraction methods); all unique extractions were run in PCR replicate (n=8 clinical replicates, 6 synthetic replicates). All samples showed expected positive (viral N1 and N2 gene targets detected with internal human RP gene control detected) or negative (N1, N2 undetermined, RP detected) results across all replicates. The average Ct values demonstrated a narrow standard deviation across both extraction and PCR replicates (Table 1 ). Further, we tested five-fold dilutions of input RNA into nuclease free water for all of these samples. Each dilution remained positive for SARS-CoV-2; and demonstrated delta Ct values ranging between 2.01-3.16 for the viral N1 and N2 targets ( Table 2 ). The internal human control RP gene target showed the greatest amount of delta Ct variability in this test.