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

We utilized the synthetic positive extraction control and a synthetic negative extraction control for validation of the lower limit of detection for the assay. These extraction controls are delivered in a synthetic transport matrix including human gDNA. We diluted 200 viral cp/µL standard control into the negative extraction control matrix containing 75 human gDNA copies/µL to achieve final viral targets of 20, 10, and 5 copies per microliter. We performed 2 to 4 extractions of aliquots at each copy number target using a combination of Qiagen and Macherey Nagel extraction kits; 2 to 5 PCR replicates were performed for each unique extraction. At 5 viral copies/µL input to extraction, we successfully detected N1 and N2 in 20 of 20 replicates (Table 8) , establishing a reliable limit of detection (LOD). The average N1 Ct at this LOD was 36.0 for the Qiagen extraction and 36.7 for the Macherey Nagel extraction; the average N2 Ct values were 35.8 and 37.2 respectively. Overall, the experiment had 97% accuracy at 1X to 2X of the limit of detection (5-10 cp/µL). One of ten PCR replicates at the 10 cp/µL input level returned an inconclusive result, detecting N1 but not N2.