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

Analytical Accuracy on Clinical Biospecimens Analytical accuracy evaluation was then performed on ten positive and ten negative residual clinical biospecimens collected by nasopharyngeal (n=7), oropharyngeal (n=1), or NP/OP combination (n=12) swabs into universal transport medium. Each sample was split into three separate 100 µL aliquots and processed independently through Qiagen (QIA), Macherey Nagel (MN), and bioMérieux easyMAG (EMAG) RNA extraction kits with 100 µL elution volumes, yielding a total of 60 extraction samples. All 30 positive and all 30 negative extraction samples were correctly assigned by the assay in comparison to orthogonal results from the outside laboratory (Table 6 ). Furthermore, the Ct values for each unique clinical sample were similar across the three different extraction methods assessed (Supplemental Table 1 ).