In-house modification and improvement of the CDC real-time PCR diagnostic assay for SARS-CoV-2 detection

  1. Srirupa Das
  2. Candice Dowell-Martino
  3. Lisa Arrigo
  4. Paul N. Fiedler
  5. Sandra Lobo

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Abstract

The world is currently facing an unprecedented pandemic caused by the novel coronavirus SARS-CoV-2 (COVID-19) which was first reported in late 2019 by China to the World Health Organization (WHO). The containment strategy for COVID-19, which has non-specific flu-like symptoms and where upwards of 80% of the affected has either mild or no symptoms, is critically centered upon diagnostic testing, tracking and isolation. Thus, the development of specific and sensitive diagnostic tests for COVID-19 is key towards the first successful step of disease management. Public health organizations like the WHO and the US-based Centers for Disease Control and Prevention (CDC) have developed real-time PCR (RT-PCR) based diagnostic tests to aid in the detection of acute infection. In this study we sought to modify the CDC RT-PCR diagnostic assay protocol to increase its sensitivity and to make the assay directly portable to health care providers in a community-based hospital setting. A number of modifications to the original protocol were tested. Increasing the RT-PCR annealing temperature by 7°C to 62°C was associated with the most significant improvement in sensitivity, wherein the cycle-threshold (Ct) value for the N2 assay was reduced by ∼3 units, in effect both reducing the overall number of inconclusive results and yielding N1/N2 assays to have similar Ct values. The limit of detection of the modified assay was also improved (0.86 RNA copies/µl for both nCoV 2019_N1/N2 assays) compared to the CDC RT-PCR diagnostic assay (1 and 3.16 RNA copies/µl for nCoV 2019_N1 and N2 assay, respectively). Using this modification, there was no significant effect on SARS-CoV-2 detection rate when viral RNA extraction was performed either manually or through an automated extraction method. We believe this modified protocol allows for more sensitive detection of the virus which in turn will be useful for pandemic management.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.10.20150771: (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.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Sample preparation and viral RNA extraction for LOD determination: LOD studies of SARS-CoV-2 were performed using a quantitative synthetic SARS-CoV-2 RNA (ATCC, Cat No. VR-3276SD) that was spiked into a diluent consisting of a suspension of human A549 cells (500 cells) and 140µl of VTM, to mimic a positive clinical specimen.
    A549
    suggested: None

    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:
    False negative results or limitations of PCR-based diagnostic tests can lead to positive COVID-19 patients being diagnosed otherwise, discharged or allowed to leave quarantine and move freely in the society, aiding in transmission of the disease. Even the most widely used point-of-care test in the US, the Abbott ID Now (based on real-time PCR), has been reported to only have an accuracy of ∼85% which led to FDA-issued alerts regarding false negatives [26]. Thus, it is a necessity to derive rapid tests that are not only specific, but also highly sensitive. In this study we modified the CDC real-time PCR diagnostic assay for COVID-19 in an effort to improve the performance with regard to sensitivity for viral detection and to allow our direct implementation of this tool in a community-based hospital setting. In addition to the list of pathogenic agents that the CDC tested for specificity, we extended the specificity testing to include an additional 27 clinically relevant human pathogenic microbial agents (Supplemental Table 1) and identified no cross-reactivity with any. Modification of the PCR annealing temperature facilitated the development of a more robust assay by lowering the Ct values of the N2 assay in particular (Table 3). An increase in the annealing temperature to 62°C from the CDC recommended 55°C, allowed the N1 and N2 assays to yield similar Ct values that were much lower than the cut-off value of 40 that the CDC recommends as being read as a positive result [5]. ...

    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

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  2. Version published to 10.1101/2020.07.10.20150771v1 on medRxiv