Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays
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Abstract
Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings.
Methods
We evaluated a guanidium thiocyanate-based buffer, eNAT ™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C.
Results
SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log 10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions.
Conclusion
eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.
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SciScore for 10.1101/2021.01.15.21249891: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethical considerations: The use of saliva from confirmed COVID-19 negative volunteers was approved by Rutgers Institutional Review Board, IRB# 2020001786. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Calculating eNAT sterilizing activity: Viral cytopathic effect (CPE) was determined in all samples by both direct infection in Vero E6 cell lines in 6-well plates and TCID50 assay by serial diluting of the samples on 96-well plates. Vero E6suggested: NoneSoftware and Algorithms Sentences Resou… SciScore for 10.1101/2021.01.15.21249891: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethical considerations: The use of saliva from confirmed COVID-19 negative volunteers was approved by Rutgers Institutional Review Board, IRB# 2020001786. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Calculating eNAT sterilizing activity: Viral cytopathic effect (CPE) was determined in all samples by both direct infection in Vero E6 cell lines in 6-well plates and TCID50 assay by serial diluting of the samples on 96-well plates. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Statistical analysis: Standard statistical analyses (average, standard deviation, and ANOVA) were performed using GraphPad Prism 8.4.3 for Windows. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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