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Development and Analytical Validation of a One-Step Five-Plex RT-ddPCR Assay for the Quantification of SARS-CoV-2 Transcripts in Clinical Samples

  • Areti Strati*
    Areti Strati
    Lab of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, 15771 Athens, Greece
    *Email: [email protected]
    More by Areti Strati
  • Martha Zavridou
    Martha Zavridou
    Lab of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, 15771 Athens, Greece
  • Dimitrios Paraskevis
    Dimitrios Paraskevis
    Department of Hygiene, Epidemiology and Medical Statistics, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece
  • Gkikas Magiorkinis
    Gkikas Magiorkinis
    Department of Hygiene, Epidemiology and Medical Statistics, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece
  • Spyros Sapounas
    Spyros Sapounas
    National Public Health Organization, 15123 Athens, Greece
  • Pagona Lagiou
    Pagona Lagiou
    Department of Hygiene, Epidemiology and Medical Statistics, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece
  • Nikolaos S. Thomaidis
    Nikolaos S. Thomaidis
    Lab of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, 15771 Athens, Greece
  • , and 
  • Evi S. Lianidou*
    Evi S. Lianidou
    Lab of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, 15771 Athens, Greece
    *Email: [email protected]
Cite this: Anal. Chem. 2022, 94, 36, 12314–12322
Publication Date (Web):August 12, 2022
https://doi.org/10.1021/acs.analchem.2c00868
Copyright © 2022 American Chemical Society

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    Abstract

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    Highly sensitive methodologies for SARS-CoV-2 detection are essential for the control of COVID-19 pandemic. We developed and analytically validated a highly sensitive and specific five-plex one-step RT-ddPCR assay for SARS-CoV-2. We first designed in-silico novel primers and probes for the simultaneous absolute quantification of three different regions of the nucleoprotein (N) gene of SARS-CoV-2 (N1, N2, N3), a synthetic RNA as an external control (RNA-EC), and Beta-2-Microglobulin (B2M) as an endogenous RNA internal control (RNA-IC). The developed assay was analytically validated using synthetic DNA and RNA calibrator standards and then was applied to 100 clinical specimens previously analyzed with a commercially available CE-IVD RT-qPCR assay. The analytical validation of the developed assay resulted in very good performance characteristics in terms of analytical sensitivity, linearity, analytical specificity, and reproducibility and recovery rates even at very low viral concentrations. The simultaneous absolute quantification of the RNA-EC and RNA-IC provides the necessary metrics for quality control assessment. Direct comparison of the developed one-step five-plex RT-ddPCR assay with a CE-IVD RT-qPCR kit revealed a very high concordance and a higher sensitivity [concordance: 99/100 (99.0%, Spearman’s correlation coefficient: −0.850, p < 0.001)]. The developed assay is highly sensitive, specific, and reproducible and has a broad linear dynamic range, providing absolute quantification of SARS-COV-2 transcripts. The inclusion of two RNA quality controls, an external and an internal, is highly important for standardization of SARS-COV-2 molecular testing in clinical and wastewater samples.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.2c00868.

    • Materials and Methods, one-step RT-ddPCR, recovery (%) and RSD (%) obtained from one-step RT-ddPCR analysis of clinical samples (n = 100) spiked with 4000 copies (200 copies/μL) of RNA-external control; and Kruskal–Wallis test for the comparison of the distribution of N1, N2, and N3 absolute number of copies among 81 clinical samples (PDF)

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