Abstract
We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as ‘direct to PCR’ assays that do not involve RNA extraction or chemical neutralisation methods.
Highlights
Coronavirus disease (COVID-19) is caused by a novel coronavirus, SARS-CoV-2, that emerged in Wuhan City, China in December 2019
murine hepatitis virus (MHV) is a prototype of the group II coronavirus in common with severe acute respiratory syndrome coronaviruses such as SARS-CoV-218
Sequencing quality was not changed by the bulk heat inactivation (HI) workflow, as illustrated by an equivalent base coverage prior to and following implementation of the bulk HI protocol at the Cambridge COVID-19 Testing Centre (CCTC) (Supplementary Fig. S11). This is the first report of a high-throughput HI protocol for SARS-CoV-2 testing that is performed on UN733 packaged OP/NP swab samples in bulk
Summary
Coronavirus disease (COVID-19) is caused by a novel coronavirus, SARS-CoV-2, that emerged in Wuhan City, China in December 2019. The approved CCTC process detects the SARS-Cov-2 ORF1ab gene by RT-qPCR in RNA extracted from clinical OP/NP swab samples. In this standard protocol, sample tubes carrying potentially viable SARSCov-2 were removed from their secondary containment, checked and transferred into sample racks. Viral inactivation occurs at the point of lysis buffer addition, as a result of its guanidine isothiocyanate content, followed by an additional heating step of 65 °C for 10 min in a small dry air laboratory oven for inactivation of any viral material on the sample plate outer surfaces These steps are manually performed within a microbiological safety cabinet (MSC) in a Containment Level 2 laboratory. We describe a significant change to the standard workflow by the development of a heat inactivation (HI) protocol for clinical OP/NP swab samples within their original UN3373 secondary containment that enables the HI of 500 swab samples/hour per oven and all subsequent laboratory processing on the open bench
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