RT-qPCR-based tests for SARS-CoV-2 detection in pooled saliva samples for massive population screening to monitor epidemics

  1. Michał Różański
  2. Aurelia Walczak-Drzewiecka
  3. Jolanta Witaszewska
  4. Ewelina Wójcik
  5. Arkadiusz Guziński
  6. Bogumił Zimoń
  7. Rafał Matusiak
  8. Joanna Kazimierczak
  9. Maciej Borowiec
  10. Katarzyna Kania
  11. Edyta Paradowska
  12. Jakub Pawełczyk
  13. Jarosław Dziadek
  14. Jarosław Dastych

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Abstract

Swab, RT-qPCR tests remain the gold standard of diagnostics of SARS-CoV-2 infections. These tests are costly and have limited throughput. We developed a 3-gene, seminested RT-qPCR test with SYBR green-based detection designed to be oversensitive rather than overspecific for high-throughput diagnostics of populations. This two-tier approach depends on decentralized self-collection of saliva samples, pooling, 1 st -tier testing with highly sensitive screening test and subsequent 2 nd -tier testing of individual samples from positive pools with the IVD test. The screening test was able to detect five copies of the viral genome in 10 µl of isolated RNA with 50% probability and 18.8 copies with 95% probability and reached Ct values that were highly linearly RNA concentration-dependent. In the side-by-side comparison, the screening test attained slightly better results than the commercially available IVD-certified RT-qPCR diagnostic test DiaPlexQ (100% specificity and 89.8% sensitivity vs. 100% and 73.5%, respectively). Testing of 1475 individual clinical samples pooled in 374 pools of four revealed 0.8% false positive pools and no false negative pools. In weekly prophylactic testing of 113 people within 6 months, a two-tier testing approach enabled the detection of 18 infected individuals, including several asymptomatic individuals, with substantially lower cost than individual RT-PCR testing.

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  1. Version published to 10.1038/s41598-022-12179-4
  2. ScreenIT

    SciScore for 10.1101/2021.05.06.21256733: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: , Proteon Pharmaceuticals S.A. in the study was approved by the Bioethical Committee at the University of Lodz (Res. nr 3/KBBN-UŁ/I/2020-21).
    Consent: Written informed consent was obtained from all participants before study entry.
    Sex as a biological variablenot detected.
    RandomizationEach positive sample was pooled by combining 3, 5 or 7 randomly chosen negative samples.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Subsequently, the cost of the screening test was calculated based on the following premises: 1) positive cases were randomly distributed among the tested population with an even distribution; 2) the test had ideal sensitivity and specificity; and 3) if the pool provided a positive result, each sample within the pool was subsequently tested individually with reference, validated, diagnostic tests.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Samples of genomic RNA isolated from human coronaviruses HCoV 229E and HCoV NL63 and respiratory syncytial viruses RSV A2001/3-12 and RSV B1 were obtained from BEI Resources.
    HCoV NL63
    suggested: None
    SARS-CoV-2 RNA isolated from infected Vero cell culture was serially diluted in ultrapure water.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Software and Algorithms
    SentencesResources
    Using BLAST 2.9.0+ (default parameters) and reference gene sequences as queries, desired genes were extracted from the remaining SARS-CoV-2 genomes.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    Curve fitting was performed in GraphPad Prism software.
    GraphPad Prism
    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: 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.

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

    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.

  3. Version published to 10.1101/2021.05.06.21256733v1 on medRxiv