Redesigning SARS-CoV-2 clinical RT-qPCR assays for wastewater RT-ddPCR

  1. Raul Gonzalez
  2. Allison Larson
  3. Hannah Thompson
  4. Errin Carter
  5. Xavier Fernandez Cassi

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Abstract

COVID-19 wastewater surveillance has gained widespread acceptance to monitor community infection trends. Wastewater samples primarily differ from clinical samples by having low viral concentrations due to dilution, and high levels of PCR inhibitors. Therefore, wastewater samples should be processed by appropriately designed and optimized molecular workflows to accurately quantify targets. Digital PCR has shown to be more sensitive and resilient to environmental matrix inhibition. However, most SARS-CoV-2 assays have been designed for clinical use on RT-qPCR instruments, then adopted to digital PCR platforms. But it is unknown whether clinical RT-qPCR assays are adequate to use on digital PCR platforms. Here we designed an N and E gene multiplex (ddCoV_N and ddCoV_E) specifically for RT-ddPCR and benchmarked them against the nCoV_N2 and E_Sarbeco assays. ddCoV_N and ddCoV_E have equivalent limits of detections and wastewater sample concentrations to NCoV_N2 and E_Sarbeco but showed improved signal-to-noise ratios that eased interpretation and ability to multiplex. From GISAID downloaded unique sequences analyzed, 2.12% and 0.83% present a mismatch or would not be detected by the used primer/probe combination for the ddCoV_N and ddCoV_E, respectively.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.02.21252754: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    We have designed 2 assays targeting N-and E-genes using Primer3Plus software (Untergasser et al., 2007) and the criteria found in Supplemental Table S1.
    Primer3Plus
    suggested: (Primer3Plus, RRID:SCR_003081)
    In-silico Specificity and primer/probe mismatch analysis: BLASTn (Altschul et al., 1990) was used to test the in-silico specificity of the ddCoV_N and ddCoV_E primers against viruses (taxid: 10239) and bacteria (taxid: 2) in the nr database.
    BLASTn
    suggested: (BLASTN, RRID:SCR_001598)
    Using Geneious software, newly designed primers/probes for the nucleocapsid protein (ddCoV_N) and the envelope gene (ddCoV_E) were mapped using Geneious primer mapper against the retrieved sequences allowing a maximum of 3 mismatches.
    Geneious
    suggested: (Geneious, RRID:SCR_010519)

    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.

    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 checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.03.02.21252754 on medRxiv