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

. CC-BY-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.02.022186 doi: bioRxiv preprint 20 ul reaction volume comparisons For 20 µL reaction volume comparison, the reactions were scaled up uniformly from the 10 µL volume. Three separate 20 µL RT-qPCR reactions were set up in a 384-well Barcoded plate (Thermo Scientific) for either the N1, N2, or RP primers and probes. 5 µL extracted RNA was added to 15 µL qPCR mastermix comprised of the following components: 3.1 µL water 10 µL GoTaq ® Probe qPCR Master Mix with dUTP (2X) (Promega, Cat # A6120 and A6121) 0.4 µL GoScriptTM RT Mix for 1-Step RT-qPCR (Promega, Cat # A6120 and A6121) 1.5 µL primer/probe sets for either N1, N2, or RP (IDT) qPCR cycling conditions Reactions were cycled in a QuantStudio QS5 (ThermoFisher) for one cycle of 45°C for 15 minutes, followed by one cycle of 95°C for 2 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. A minimum of two no template controls (NTCs) were included on all runs. Baselines were allowed to calculate automatically, and a ΔRn threshold of 0.5 was selected and set uniformly for all runs. Ct values were exported and analyzed in Microsoft Excel. Amplification curves were manually reviewed.