Rapid detection of SARS-CoV-2 and other respiratory viruses by using LAMP method with Nanopore Flongle workflow

  1. Jingjing Li
  2. Weipeng Quan
  3. Shuge Yan
  4. Shuangju Wu
  5. Jianhu Qin
  6. Tingting Yang
  7. Fan Liang
  8. Depeng Wang
  9. Yu Liang

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Abstract

The ongoing novel coronavirus (COVID-19) outbreak as a global public health emergency infected by SARC-CoV-2 has caused devastating loss around the world. Currently, a lot of diagnosis methods have been used to detect the infection. The nucleic acid (NA) testing is reported to be the clinical standard for COVID-19 infection. Evidence shows that a faster and more convenient method to detect in the early phase will control the spreading of SARS-CoV-2. Here, we propose a method to detect SARC-Cov-2 infection within two hours combined with Loop-mediated Isothermal Amplification (LAMP) reaction and nanopore Flongle workflow. In this approach, RNA reverse transcription and nucleic acid amplification reaction with one step in 30 minutes at 60-65°C constant temperature environment, nanopore Flongle rapidly adapter ligated within 10 minutes. Flongle flow cell sequencing and analysis in real-time. This method described here has the advantages of rapid amplification, convenient operation and real-time detection which is the most important for rapid and reliable clinical diagnosis of COVID-19. Moreover, this approach not only can be used for SARS-CoV-2 detection but also can be extended to other respiratory viruses and pathogens.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.03.131474: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationThe FRA is a barcoded transposome complex, which can cleave randomly DNA and add barcoded transposase adapters to the cleavage sites.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    All primers were aligned to the published SARS-CoV-2 sequences and others respiratory using BLAST.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    All the dilution gradient and pure water (negative control) were amplified by LAMP.
    LAMP
    suggested: (LAMP, RRID:SCR_001740)
    To test the limit of detection, the amplification products of dilution gradient 3.25×10^4, 3.25×10^3, 1.1 × 10^3, 6.5 × 10^2, 3.25×10^2 copies/mL and negative control total 12 samples were constructed another barcoding library (Oxford Nanopore, SQK-RBK004) as described above and sequenced using a PromethION flowcell to achieve more data.
    PromethION
    suggested: (PromethION, RRID:SCR_017987)
    Quality control was processed by MinKNOW (version 3.6.5) software pipeline in the machine with reads quality value score cutoff 7.
    MinKNOW
    suggested: None
    We align nanopore long reads to virus genome database with minimap2 (version 2.1) and use samtools (version 1.9) to demonstrate the coverage and depth.
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)

    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.
    • 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 10.1101/2020.06.03.131474v1 on bioRxiv