Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction
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Abstract
Convenient, repeatable, large-scale molecular testing for SARS-CoV-2 would be a key weapon to help control the COVID-19 pandemic. Unfortunately, standard SARS-CoV-2 testing protocols are invasive and rely on numerous items that can be subject to supply chain bottlenecks, and as such are not suitable for frequent repeat testing. Specifically, personal protective equipment (PPE), nasopharyngeal (NP) swabs, the associated viral transport media (VTM), and kits for RNA isolation and purification have all been in short supply at various times during the COVID-19 pandemic. Moreover, SARS-CoV-2 is spread through droplets and aerosols transmitted through person-to-person contact, and thus saliva may be a relevant medium for diagnosing SARS-CoV-2 infection status. Here we describe a saliva-based testing method that bypasses the need for RNA isolation/purification. In experiments with inactivated SARS-CoV-2 virus spiked into saliva, this method has a limit of detection of 500-1000 viral particles per mL, rivalling the standard NP swab method, and initial studies also show excellent performance with 100 clinical samples. This saliva-based process is operationally simple, utilizes readily available materials, and can be easily implemented by existing testing sites, thus allowing for high-throughput, rapid, and repeat testing of large populations.
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SciScore for 10.1101/2020.06.18.159434: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding In all studies conducted, researchers were blinded to the results obtained from clinical RT-qPCR tests performed on NP swabs at the Carle Foundation Hospital. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Cycle threshold (Ct) values were plotted as single replicate values on a scatter plot, using GraphPad Prism 8 (version 8.4.2). 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…
SciScore for 10.1101/2020.06.18.159434: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding In all studies conducted, researchers were blinded to the results obtained from clinical RT-qPCR tests performed on NP swabs at the Carle Foundation Hospital. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Cycle threshold (Ct) values were plotted as single replicate values on a scatter plot, using GraphPad Prism 8 (version 8.4.2). 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: We detected the following sentences addressing limitations in the study:Direct saliva-to-RT-qPCR process, key advances and remaining limitations: The direct saliva-to-RT-qPCR method described herein, bypassing NP swabs, VTM, and RNA isolation/purification, was enabled by a handful of key discoveries. First, the time and duration of heating the saliva sample is critical. Standard protocols for heat inactivation of SARS-CoV-2 call for heating at ~60°C for 30 minutes;30,31 while these conditions inactivate the virus, they do not allow for successful SARS-CoV-2 detection via direct RT-qPCR, likely because of the persistence of as-yet-unidentified factors in saliva that are inhibitory to RT-qPCR. Heating at 95°C for 30 minutes likely inactivates these inhibitory components and allows for excellent SARS-CoV-2 detection in this direct process that bypasses RNA isolation/purification. Second, while TE buffer performs well, consistent with another report successfully using TE to extract dry NP swabs,17 TBE buffer provides more reliability and consistency in our direct saliva-to-RT-qPCR detection of SARS-CoV-2. Finally, the addition of the non-ionic detergent Tween-20 also helped improve detection of SARS-CoV-2, possibly by facilitating the opening of the viral capsid to allow the release of RNA to provide sufficient template for RT-qPCR detection. Our preliminary assessment of clinical samples is very promising, especially given that these samples were not collected and processed under the optimized protocol (they were collected before our discovery of th...
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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SciScore for 10.1101/2020.06.18.159434: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding In all studies conducted , researchers were blinded to the results obtained from clinical RT-qPCR tests performed on NP swabs at the Carle Foundation Hospital. 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 In order to validate the specificity of our detection system to SARS-CoV-2 , saliva was spiked with or without SARS-CoV-2 ( γ-irradiated virus , synthetic N-transcript) , two other human coronaviruses ( OC43 , 229E) , SARS and MERS synthetic RNA , and human RNA ( extracted … SciScore for 10.1101/2020.06.18.159434: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding In all studies conducted , researchers were blinded to the results obtained from clinical RT-qPCR tests performed on NP swabs at the Carle Foundation Hospital. 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 In order to validate the specificity of our detection system to SARS-CoV-2 , saliva was spiked with or without SARS-CoV-2 ( γ-irradiated virus , synthetic N-transcript) , two other human coronaviruses ( OC43 , 229E) , SARS and MERS synthetic RNA , and human RNA ( extracted from HEK 293 cells) . HEK 293suggested: CLS Cat# 300192/p777_HEK293, CVCL_0045Software and Algorithms Sentences Resources In the future , development of analogous saliva-based processes that bypass RNA isolation/purification can be envisioned for alternative back-end detection technologies , such as the LAMP method,2,47 which if successful would result in an even shorter overall time from sample collection to results . LAMPsuggested: (LAMP, SCR_001740)Cycle threshold (Ct) values were plotted as single replicate values on a scatter plot, using GraphPad Prism 8 (version 8.4.2). GraphPad Prismsuggested: (GraphPad Prism, SCR_002798)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 OddPub: Thank you for sharing your data.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.
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