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

When handling clinical samples prior to viral inactivation, we use standard practices for biosafety level 2, with enhanced personal protective equipment (BSL2+). This includes a disposable gown, face protection with either full shield or mask with eye protection, Tyvek sleeves, double gloves, and a clean room tacky mat located at the BSL2+ room's threshold. All work with patient samples is performed in a biological safety cabinet (BSC), including centrifugation, vortexing and heating. Additional BSL2+ precautions include: changing outer gloves after any manipulation of samples prior to removing hands from the BSC, reduced pipette speed to minimize aerosol risks, working over towels soaked in 10% bleach, and disposing pipet tips used with clinical samples into a container with bleach. For viral inactivation, clinical samples are first placed in tubes containing viral lysis buffer and treated according to manufacturer's protocol. Samples are transferred into a new clean tube containing 96-100% ethanol as per the next step in the RNA extraction protocol. After inactivation, sample tubes are surface decontaminated, placed in secondary containment, and moved into a separate room where the remainder of the RNA extraction protocol is carried out at standard BSL2 practices in a BSC. Personal protective equipment for RNA extraction include gloves, gowns and eye protection.