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

Using a combination of Emergency Use Authorized (EUA) and research use only (RUO) reagents, our multi-disciplinary clinical science team was able to validate analytic performance of this SARS-CoV2 detection assay in a very short time frame (5 days) and in the face of extraordinary barriers to the acquisition of EUA-cleared reagents. Our results demonstrate robust performance of a 10 µL volume, 384-well format qRT-PCR on the QuantStudio 5 instrument using a RUO Promega one-step mastermix. This format will support test throughput and increase the number of reactions possible in the current landscape of scarce reagents. Our results also demonstrate suitable performance of the Macherey-Nagel Nucelospin Virus extraction kit in comparison to two other EUA methods. We developed robust biosafety specimen handling procedures to ensure safety of all laboratory staff and a custom informatics system to promote scalability of sample accessioning, testing, and resulting.