Design of a Novel Multiplex Real Time RT-PCR Assay for SARS-CoV-2 Detection

  1. Wei Zhen
  2. Gregory J. Berry

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Abstract

Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 386,000 deaths globally as of June 4, 2020. In this study, we developed a novel multiplex real time reverse transcription (RT)-PCR test for detection of SARS-CoV-2, with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) of SARS-CoV-2 and a hydrolysis Taqman probe designed to specifically detect SARS-CoV-2. Following our design, we evaluated the Limit of detection (LOD) and clinical performance of this laboratory-developed test (LDT). A LOD study with inactivated whole virus exhibited equal performance to that seen in the modified CDC assay with a final LOD of 1,301 ± 13 genome equivalents/ml for our assay vs 1,249 ± 14 genome equivalents/ml for the modified CDC assay. In addition, a clinical evaluation with 270 nasopharyngeal (NP) swab specimens exhibited 98.5% positive percent agreement and 99.3% negative percent agreement with the modified CDC assay. The multiplex design of this assay allows the testing of 91 patients per plate, versus a maximum of 29 patients per plate on the modified CDC assay, providing the benefit of testing significantly more patients per run and saving reagents during a time when both of these parameters have been critical. Our results demonstrate that our multiplex assay performs as well as the modified CDC assay, but is more efficient and cost effective and is therefore adequate for use as a diagnostic assay and for epidemiological surveillance and clinical management of SARS-CoV-2.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.04.135608: (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
    Primers and probe design: Available whole genome sequence of SARS-CoV-2 (as of February 27, 2020) retrieved from the NCBI GenBank database and the Global Initiative on Sharing All Influenza Database (GISAID) were aligned using Clustal Omega software from EMBL-EBI.
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)

    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:
    Limitations of our study include that our LDT is a single site evaluation at Northwell Health Laboratories. In addition, we only use a single target gene for SARS-CoV-2 detection. While there has been a trend toward dual-target design in commercial assay for the detection of pathogens (8,12), occasional monitoring of SARS-CoV-2 sequences to verify that mutations have not developed in the region targeted by our primers and probe is an adequate quality monitor to ensure continued consistent analytical performance. In summary, our LDT has comparable analytical sensitivity and accuracy for specific detection of SARS-CoV-2 RNA when compared to the modified CDC assay. In addition, it also showed superior efficiency and cost-effectiveness and can be somewhat easily-established in other laboratories. These findings make our novel multiplex SARS-CoV-2 assay a suitable alternative for the accurate diagnosis of SARS-CoV-2, with the added benefit of superior efficiency and cost-effectiveness.

    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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • No funding statement was detected.
    • 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 10.1101/2020.06.04.135608v1 on bioRxiv