Qualitative and quantitative effects of thermal treatment of naso-oropharyngeal samples before cobas SARS-CoV-2 testing

Abstract

To improve laboratory safety we thermally treated naso-oropharyngeal samples before testing with the cobas SARS-CoV-2 assay. This study aimed to determine if thermal treatment significantly affects the qualitative detection of SARS-CoV-2 and the quantitative measurement of cobas SARS-CoV-2 ORF1a and E-gene target copy number using an in-house quantitative method. A collection of positive ( n= 238) and negative samples ( n= 196) was tested in parallel comparing thermal treatment (75°C for 15 minutes) to room-temperature. There were no significant differences in the final qualitative outcomes for thermal treatment versus room-temperature (99.8% agreement) despite a statistically significant reduction (p<0.05) in target copy number following thermal treatment. The median ORF1a and E-gene reduction in target copy number was ­0.07 (1.6%) and ­0.22 (4.2%) log 10 copies/mL respectively. The standard curves for both ORF1a and E-gene targets were highly linear (r 2 =0.99). Good correlation was observed for ORF1a (r 2 =0.96) and E-gene (r 2 =0.98) comparing thermal treatment to room-temperature control.

Improving the efficiency and biosafety of respiratory syncytial virus identification using a nucleic acid extraction‐free reagent

“…The conventional VTM reagent allows viruses to remain viable; the resulting high risk of infection during transportation and detection leads to increased costs because of the need for greater protection. The use of heat to inactivate viruses in clinical samples can affect the stability of viral nucleic acids, particularly RNA 22 . Sampling solutions that contain guanidine salts can effectively inactivate viruses, but a nucleic acid extraction step is required for sample detection.…”

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

“…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 ).…”

“…During the early stages of the pandemic, samples were tested using the cobas SARS-CoV-2 assay (Roche catalog number 09175431190) as the primary testing method. Thermal pretreatment of the sample was performed before cobas testing ( 3 , 4 ). We implemented the PlexPCR SARS-CoV-2 assay (SpeeDx catalog number 1301384) in September 2020 as an additional testing method to increase testing capacity ( 5 ).…”

“…These tools are the strategic cornerstone to mitigate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread, facilitating early diagnosis, isolation of infected individuals, and clearance of essential personnel to continue to work ( 2 ). Since 20 March 2020, we have performed more than 700,000 tests using two commercial extraction and detection systems: the cobas SARS-CoV-2 assay (Roche Molecular Systems, Branchburg, NJ, USA), and the PlexPCR SARS-CoV-2 assay (SpeeDx, Eveleigh, NSW, Australia) ( 3 – 6 ). The PlexPCR SARS-CoV-2 assay workflow in our laboratory utilizes a maximum of four MagNA Pure 96 instruments (Roche) for RNA extraction, two PlexPrep liquid handlers (SpeeDx) for 384-well PCR plate preparation, and four LightCycler 480 thermal cyclers (Roche) for amplification and detection.…”