Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing
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Abstract
The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.
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SciScore for 10.1101/2021.04.29.21256345: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources A new 1 µL inoculation loop was then used to sample the inoculated RT-qPCR reaction mixture and introduced into the culture media of Vero-E6 cells seeded in a 6-well culture plate. Vero-E6suggested: NoneSoftware and Algorithms Sentences Resources Fluorescent VSV inactivation assay: Sodium dodecyl sulfate (SDS; Sigma Aldrich, 436143) inactivation was performed by dipping a 1 µL inoculation loop (Globe Scientific, 2810) in a VSV-eGFP-SARS-CoV-2 suspension at 107 … SciScore for 10.1101/2021.04.29.21256345: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources A new 1 µL inoculation loop was then used to sample the inoculated RT-qPCR reaction mixture and introduced into the culture media of Vero-E6 cells seeded in a 6-well culture plate. Vero-E6suggested: NoneSoftware and Algorithms Sentences Resources Fluorescent VSV inactivation assay: Sodium dodecyl sulfate (SDS; Sigma Aldrich, 436143) inactivation was performed by dipping a 1 µL inoculation loop (Globe Scientific, 2810) in a VSV-eGFP-SARS-CoV-2 suspension at 107 pfu/mL and applying the inoculation loop to a dried 1 µL spot of SDS/TE (0.1% w/v SDS, Tris-HCl, pH 8.0) on the side wall of a PCR reaction tube, followed by mixing for 15-120 seconds. SDS; Sigma Aldrichsuggested: NoneResults 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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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.
Results from scite Reference Check: We found no unreliable references.
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