Rapid and High-Throughput Reverse Transcriptase Quantitative PCR (RT-qPCR) Assay for Identification and Differentiation between SARS-CoV-2 Variants B.1.1.7 and B.1.351

  1. Oran Erster
  2. Ella Mendelson
  3. Virginia Levy
  4. Areej Kabat
  5. Batya Mannasse
  6. Hadar Asraf
  7. Roberto Azar
  8. Yaniv Ali
  9. Rachel Shirazi
  10. Efrat Bucris
  11. Dana Bar-Ilan
  12. Orna Mor
  13. Michal Mandelboim
  14. Danit Sofer
  15. Shai Fleishon
  16. Neta S. Zuckerman

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Abstract

This study describes the design and utilization of a multiplex reverse transcriptase quantitative PCR (RT-qPCR) to identify SARS-COV-2 (SC2) RNA in general and, specifically, to detect whether it is of lineage B.1.1.7 or B.1.351. Implementation of this method in diagnostic and research laboratories worldwide may help the efforts to contain the COVID-19 pandemic.

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  1. Version published to 10.1128/spectrum.00506-21
  2. ScreenIT

    SciScore for 10.1101/2021.05.19.21257439: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Design of VOC-specific qPCR reactions: Analysis of SC-2 sequences and primer simulations were performed using the Geneious software package (https://www.geneious.com/) and the NCBI BLAST analysis tools
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    The reaction data were analyzed using the Bio-Rad CFX Maestro software.
    Bio-Rad CFX Maestro
    suggested: None
    CFX
    suggested: None
    Next Generation Whole genome sequencing of clinical samples: COVID-seq kit was used for library preparation as per manufacturer’s instructions (Illumina, https://www.illumina.com/).
    https://www.illumina.com/
    suggested: (Illumina, RRID:SCR_010233)
    Library validation and mean fragment size was determined by TapeStation 4200 via DNA HS D1000 kit (Agilent, https://www.agilent.com/).
    https://www.agilent.com/
    suggested: (Agilent Technologies, RRID:SCR_013575)
    Bioinformatic and phylogenetic analysis: Fastq files underwent quality control using FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/) and MultiQC [10] and low-quality sequences were filtered using trimmomatic [PMID: 24695404].
    Bioinformatic
    suggested: (QFAB Bioinformatics, RRID:SCR_012513)
    FastQC
    suggested: (FastQC, RRID:SCR_014583)
    MultiQC
    suggested: (MultiQC, RRID:SCR_014982)
    trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    Mapping to SARS-CoV-2 genome (NC_045512.2) was performed with BWA mem [PMID: 19451168].
    BWA
    suggested: (BWA, RRID:SCR_010910)
    SAMtools suite [PMID: 19505943] was used to filter unmapped reads, sort and index bam files.
    SAMtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    Sequences were aligned with the SARS-CoV-2 reference sequence (NC_045512.2) with MAFFT [11] and mutation analysis was done with a custom R code using Bioconductor package seqinr [citation info in: https://cran.r-project.org/web/packages/seqinr/citation.html].
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Bioconductor
    suggested: (Bioconductor, RRID:SCR_006442)

    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.
    • 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.

    Results from scite Reference Check: We found no unreliable references.

    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.

  3. Version published to 10.1101/2021.05.19.21257439v1 on medRxiv