Heat inactivation of clinical COVID-19 samples on an industrial scale for low risk and efficient high-throughput qRT-PCR diagnostic testing

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.

Figure 1

Validation of the industrial steam oven for bulk HI. (a) The Electrolux Skyline Combi Oven and the trolley loading system. The oven is shown in temperature validation mode with thermocouple wires in place. Under normal operation these are not included. (b) Thermocouple swab vials used for measuring temperature at the point of the swab to determine exposure of potential viable virus to heat. (c) An example heat mapping study showing the thermal profile of 20 thermocouple swab vials placed throughout the oven, 2 in each of 10 trays, and including 250 samples in total to mimic a full oven. Each coloured line represents an individual thermocouple swab vial (d) Average of three heat mapping data sets of 20 thermocouple, including that shown in (c). Vertical bars represent the temperature range shown for every 10th data point. (e) Inactivation of the SARS-CoV-2 surrogate virus MHV-A59 in the Electrolux Skyline Combi Oven using the temperature profile shown in (c) and (d), showing reduction in infectivity in human cells. Data is plotted as mean + /− standard deviation. The dashed line represents the limit of detection of the assay. Data was plotted using R (v4.1.0 “Camp Pontanezen”) with ggplot2 (v3.3.3) and the figure was assembled using Affinity Designer (1.9.3).

Figure 2

SARS-CoV-2 detection by RT-qPCR following heat treatment. (a) Impact on SARS-CoV-2 RT-qPCR Cq value in OP/NP samples following incubation at 70 °C and 80 °C compared to 65 °C for 10 min. (b) Selected samples from (a) showing the effect is not Cq dependent. (c) Impact on SARS-CoV-2 RT-qPCR Cq value in OP/NP samples following incubation at 65 °C for 20, 60 and 90 min compared to 10 min. (d) Selected samples from (c) showing the effect is not Cq dependent. See Supplementary Tables 2 and 3 for summary statistics. Data was plotted using R (v4.1.0 “Camp Pontanezen”) with ggplot2 (v3.3.3) and the figure was assembled using Affinity Designer (1.9.3).

Figure 3

Bulk HI concordance data using clinical OP/NP samples. (a) Concordance of SARS-CoV-2 RT-qPCR test results for clinical OP/NP samples which tested positive in either the standard clinical assay or using the bulk HI protocol, and binned according to the clinical assay Cq. (b) Distribution of change in Cq value (Clinical Cq—Experimental Cq) versus the clinical test for the primary bulk HI protocol (bulk HI) and variations (deliberate delay of 10 or 24 h post HI, or the fall-back method of a second HI step in the lab (2HI). (c) Selected samples from (a) showing the impact of bulk HI on RT-qPCR detection is not Cq dependent. Data was plotted using R (v4.1.0 “Camp Pontanezen”) with ggplot2 (v3.3.3) and the figure was assembled using Affinity Designer (1.9.3).