Ultra-fast one-step RT-PCR protocol for the detection of SARS-CoV-2

  1. Ehsan Asghari
  2. Anna Höving
  3. Paula van Heijningen
  4. Annika Kiel
  5. Angela Krahlemann-Köhler
  6. Melanie Lütkemeyer
  7. Jonathan Storm
  8. Tanja Vollmer
  9. Cornelius Knabbe
  10. Barbara Kaltschmidt
  11. Gert de Vos
  12. Christian Kaltschmidt

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Abstract

The COVID-19 pandemic resulted in lockdowns all over the world thus affecting nearly all aspects of social life and also had a huge impact on global economies. Since vaccines and therapies are still not available for the population, prevention becomes desperately needed. One important aspect for prevention is the identification and subsequent isolation of contagious specimens. The currently used methods for diagnostics are time consuming and also hindered by the limited availability of reagents and reaction costs, thus presenting a bottle neck for prevention of COVID-19 spread. Here, we present a new ultra-fast test method which is ten times faster than conventional diagnostic tests using real time quantitative PCR (RT-qPCR). In addition, this ultra-fast method is easy to handle as well as cost effective. We translated published SARS-CoV-2 testing protocols from the Centers of Disease Control and Prevention (Atlanta, Georgia, USA) and the Charité Berlin (Germany) to the NEXTGENPCR (NGPCR) machine and combined it with a fluorescence-based endpoint measurement. Fluorescence was measured with a commercial blue light scanner. We confirmed the NEXTGENPCR results with commercially available positive controls. In addition, we isolated RNA from SARS-CoV-2 infected patients and achieved similar results to clinical RT-qPCR assays. Here, we could show correlation between the results obtained by NEXTGENPCR and conventional RT-qPCR.

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    SciScore for 10.1101/2020.06.25.20137398: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: The need for informed consent and ethical approval was waived since all materials used were anonymous samples already analysed in routine laboratory diagnostics.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    No key resources detected.

    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:
    We therefore conclude that limitation of cycle number to 40 in endpoint PCR might be a way to avoid false positive results. In these experiments with 40 cycles PCR amplification time was 10 min. The optimization of the Drosten panel was performed using synthetic RNA (Twist Synthetic SARS-CoV-2 RNA Control 1, Twist Bioscience San Francisco), thus in contrast to the CDC panel optimization included an RT step. Comparison between annealing at 58 °C (Fig.2 D) and 61 °C (Fig.2 E) yielded no obvious difference in amplification efficiency. Like 45 cycles (Fig.2 D, E), 40 cycles (Fig.2 F) lead to comparable results. As a positive side effect, the limitation of cycles can avoid false positive results as shown above and is considerably time saving as well. Thus, the protocol of Fig. 2 F was chosen for further experiments. Taken together, total reaction time including RT and PCR amounts to 16 min using 40 cycles. According to a press release from GNA biosolutions (published at munich-startup.de), their SARS-CoV-2 test in development might take 15 min. We see an advantage in the setup used here, since standard primer panels can be used and no dedicated gold nanoparticle-coupled probes are necessary. Additionally, we tested the reproducibility of the protocol used in Fig. 2 C on five different NEXTGENPCR machines (Fig. 3 A-E). We were able to reproduce identical results on all five machines. Furthermore, we excluded the occurrence of edge effects by testing different positions on the plate...

    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 10.1101/2020.06.25.20137398v1 on medRxiv