Development and Testing of a Low-Cost Inactivation Buffer That Allows for Direct SARS-CoV-2 Detection in Saliva

  1. Brandon Bustos-Garcia
  2. Sylvia Garza-Manero
  3. Nallely Cano-Dominguez
  4. Dulce Maria Lopez-Sanchez
  5. Gonzalo Salgado-Montes de Oca
  6. Alfonso Salgado-Aguayo
  7. Felix Recillas-Targa
  8. Santiago Avila-Rios
  9. Victor Julian Valdes

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Abstract

Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places with good vaccination coverage. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing in resource-limited settings. It is therefore important to develop testing protocols that are inexpensive, fast, and sufficiently sensitive. Here, we optimized the composition of a buffer (PKTP), containing a protease, a detergent, and an RNase inhibitor, which is compatible with the RT-qPCR chemistry, allowing for direct SARS-CoV-2 detection from saliva without extracting RNA. PKTP is compatible with heat inactivation, reducing the biohazard risk of handling samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (85.7% for the N1 and 87.1% for the N2 target) and correlations (R = 0.77, p < 0.001 for N1, and R = 0.78, p < 0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to minimize the PCR reaction number per patient and further reduce costs and processing time of several samples, while maintaining diagnostic standards in favor of massive testing.

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  1. Version published to 10.3390/vaccines10050730
  2. ScreenIT

    SciScore for 10.1101/2021.11.26.21266918: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsConsent: All donors provided written informed consent for the use of remnant samples for research.
    IRB: The protocol for patient enrolment and sample donation was revised and approved by the Institutional Review Board of the INER (protocol B12-20).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    All statistical analyses were undertaken in GraphPad Prism 9.0.1 (1992-2012 GraphPad Software, Inc) or the R statistical software version 3.6.3, using the PerformanceAnalytics package (V 2.0.4) for Pearson Correlation analysis (Brian G. Peterson, 2020 #28)
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (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:
    In our hands, false positive results were virtually nonexistent when assaying two viral targets, making the PKTP buffer an excellent alternative for massive testing in saliva not only in settings where trained personnel is scarce and appropriate biosafety facilities are not easily accessible, but also where budget is a limitation and testing turnaround time is a major priority. The use of PKTP buffer could facilitate epidemiological surveillance in working environments, universities or schools, even in children where NPS testing is not ideal. The calculated cost of the PKTP buffer is less than one US dollar per sample, which in conjunction with multiplexed reactions represents a clear advantage in favor of massive SARS-CoV-2 qPCR testing using saliva samples. The trade-off between diagnostic sensitivity (especially for samples with low viral load, which have been suggested to represent low-to-zero infection risk [25]), and the overall costs, implementation feasibility and testing capacity should be considered in different contexts aiming to optimize the use of scarce human and material resources.

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.11.26.21266918v1 on medRxiv