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

The preferred method for diagnosis of coronavirus infections is by one-step quantitative reverse transcriptase PCR (qRT-PCR) [1] [2] [3] [4] . For the majority of qRT-PCR procedures, RNA must be extracted from patient samples [1, 3] . SARS-CoV-2 present in patient samples must be inactivated during the lysis step of RNA extraction for the safety of individuals working with the samples. The CDC has confirmed the use of several RNA extraction kits with external lysis buffer that is effective at inactivating SARS-CoV-2 [5] . Unfortunately, access to these kits is severely limited, hampering widespread implementation of testing. Validation of new RNA extraction kits and qRT-PCR reagents is desperately needed to ease supply constraints and to increase testing worldwide. Two of the CDC approved kits, the QIAmp Viral RNA kit (Qiagen) and EasyMag NucliSENS kit (biomérieux), have lysis buffers that contain guanidinium thiocyanate and guanidine thiocyanate, respectively. A third kit, the NucleoSpin Virus RNA/DNA extraction kit (Machery Nagel, Takara) which is not currently CDC approved, contains guanidine hydrochloride in the lysis buffer. We compared these three extraction methods and performed qRT-PCR with RUO primer-probe sets targeting the CDC approved 2019-nCoV_N1, 2019-nCoV_N2 and human RNase P (RP) sequences. Responding to current shortages in reagent availability, we developed a 384-well, lower volume qRT-PCR assay with an alternative single step master mix. Our data demonstrate all three RNA extraction kits can be used with this laboratory-developed procedure to effectively detect SARS-CoV-2 and that reducing input and qRT-PCR reaction volumes can still provide sensitivity of detection down to 5 copies of viral genome per microliter (comparable to previously validated methods).