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.

High-Throughput COVID-19 Testing of Naso-Oropharyngeal Swabs Using a Sensitive Extraction-Free Sample Preparation Method

“…Despite the loss in analytical sensitivity, the overall agreement was 99.3% combined without C q stratification of results. We note that other studies have focused on positive sample comparisons and the number of negative samples tested with extraction-free approaches has been limited or absent ( 8 , 9 , 11 , 18 ). Diagnostic PCR assays are optimized for purified nucleic acids in terms of primer-probe stringency and test performance.…”

“…Optimization experiments have shown that high temperature and short durations (95 to 98°C for 5 to 15 min) improve (reduce) C q values compared to lower temperature and longer durations (60°C for 30 min) ( 9 ). Heat inactivation prior to testing with extraction-based methods also reduces the sensitivity of SARS-CoV-2 RNA detection ( 4 , 18 , 19 ). Other investigators have shown the addition of proteinase K (55°C for 15 min) followed by heat treatment also improves C q values up to three cycles compared to heat treatment alone ( 11 ).…”

Rapid and reliable inactivation protocols for the diagnostics of emerging viruses: The example of SARS‐CoV‐2 and monkeypox virus

“…As different forms of specimens, including sputum, plasma, and stool can be handled, the methods to inactivate these different samples can vary greatly, but the most common are heat and detergents 11,14,16 . Numerous studies have even described SARS‐CoV‐2 inactivation efficacy with less traditional protocols including ophthalmic solutions or repurposed therapeutic agents 17,18 .…”

Sars-Cov-2 Inactivation by a Panel of Commercialized Buffers and by Traditional Protocols (Heat or SDS)

“…[25][26][27]. The methods to inactivate these different samples can vary greatly, but the most common are heat and detergents [20,21,[28][29][30][31]. Numerous studies have even described SARS-CoV-2 inactivation efficacy with less traditional protocols including ophthalmic solutions, repurposed therapeutic agents or ultraviolet C irradiation [28,[32][33][34].…”

“…With the unprecedented high demand of diagnostics tests performed around the world, the question of handling and processing such infectious samples remains relevant. Inactivation protocols against viruses have been regularly evaluated in the past, including with heat against influenza [29,30] and SARS-CoV 2003 [31] viruses, as well as with detergents against enveloped herpes simplex and human immunodeficiency virus [32]. Indeed, with over 426 million global COVID-19 cases diagnosed by mid-February 2022 [9], public health laboratories have commissioned incremental changes to logistics processing and staff organization.…”