Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number

Abstract

The outbreak of COVID-19 has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed with three different methods: (1) incubation with TRIzol® LS Reagent for 10 min at room temperature, (2) heating in a waterbath at 56°C for 30 min, and (3) high-temperature treatment, including 121°C autoclaving for 20 min, 100°C boiling for 20 min, and 80°C heating for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of N gene and 39.85% of ORF 1ab. For samples treated at 56°C for 30 min, the copy number of N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. Viral RNA copy number dropped by 50–66% after 80°C heating for 20 min. Nearly no viral RNA was detected after autoclaving at 121°C or boiling at 100°C for 20 min. These results indicated that inactivation reduced the quantity of detectable viral RNA and may cause false negative results especially in weakly positive cases. Thus, TRIzol is recommended for sample inactivation in comparison to heat inactivation as Trizol has the least effect on RNA copy number among the tested methods.

Quantitative In-Vitro Diagnostic NMR Spectroscopy for Lipoprotein and Metabolite Measurements in Plasma and Serum: Recommendations for Analytical Artifact Minimization with Special Reference to COVID-19/SARS-CoV-2 Samples

“… 48 Incubating samples at 56 °C for 10 min, has been shown to completely denature the nucleocapsid protein of SARS-CoV 48 and similarly heating plasma at 56 °C for 25 min reduced >4 log 10 TCID 50 /mL of MERS virus, 49 where TCID 50 represents the median tissue culture infectious dose. Since heat treatment has been recommended for viral inactivation, and there is a reported study 50 which demonstrated the generation of false negative results, we therefore compared the effect of heating on the 1 H NMR-derived lipoprotein profile, CPMG and 1D spectral data sets. Heating samples at 56 °C for 30 min caused major changes across all three spectral data sets.…”

Comparison of qualitative and quantitative analyses of COVID-19 clinical samples

“…In addition to qualitative diagnosis, quantitative detection also plays a significant role in COVID-19 diagnosis, surveillance, prevention, and control. As a technology for the absolute quantification of nucleic acids, digital polymerase chain reaction (dPCR) has also been applied to SARS-CoV-2 nucleic acid detection [4] , [5] , [6] . In a dPCR assay, the sample is first diluted and evenly partitioned into many independent reaction chambers.…”

Thermal treatment of nasopharyngeal samples before cobas SARS-CoV-2 testing

“…Higher C t values for thermally treated samples may suggest a reduction in detectable virus RNA. A recent study using a commercial qualitative method (BioGerm Medical Biotechnology, Shanghai, China) and quantitative digital PCR (TargetingOne, Beijing, China) demonstrated a drop in SARS-CoV-2 copy number by 50% to 66% after heating at 80°C for 20 minutes [ 8 ]. However, internal control test performance was not evaluated (or not included as part of the assay), and correlation with other commercial in vitro diagnostic assays or cobas is not known.…”