Discrimination of SARS-CoV-2 Omicron sub-lineages BA.1 and BA.2 using a high-resolution melting-based assay: A pilot study

  1. Akira Aoki
  2. Hirokazu Adachi
  3. Yoko Mori
  4. Miyabi Ito
  5. Katsuhiko Sato
  6. Kenji Okuda
  7. Toru Sakakibara
  8. Yoshinori Okamoto
  9. Hideto Jinno

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. As of March 2022, Omicron variant BA.2 is rapidly replacing variant BA.1. As variant BA.2 may cause more severe disease than variant BA.1, variant BA.2 requires continuous monitoring. The current study aimed to develop a novel high-resolution melting (HRM) assay for variants BA.1 and BA.2 and to determine the sensitivity and specificity of our method using clinical samples. Here, we focused on the mutational spectra at three regions in the spike receptor-binding domain (RBD; R408, G446/L452, and S477/T478) for the variant-selective HRM analysis. Each variant was identified based on the mutational spectra as follows: no mutations (Alpha variant); L452R and T478K (Delta variant); G446S and S477N/T478K (Omicron variant BA.1); and R408S and S477N/T478K (Omicron variant BA.2). Upon analysis of mutation-coding RNA fragments, the melting peaks of the wild-type fragments were distinct from those of the mutant fragments. The sensitivity and specificity of this method were determined as 100% and more than 97.5%, respectively, based on 128 clinical samples (40 Alpha, 40 Delta, 40 Omicron variants BA.1/BA.1.1, and 8 Omicron BA.2). These results suggest that this HRM-based assay is a promising screening method for monitoring the transmission of Omicron variants BA.1 and BA.2.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2022.04.11.487970: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics statement: This project was approved by the Research Ethics Committee of Meijo University (Approval number: 2020-17-2) and Aichi Prefectural Institute of Public Health (Approval number: 20E-4) and was carried out according to the Infectious Diseases Control Law of Japan.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Whole-genome sequencing was performed using a next-generation sequencer (MiSeq system; Illumina Inc., San Diego, CA, USA).
    MiSeq
    suggested: (A5-miseq, RRID:SCR_012148)

    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:
    This study needs to be interpreted in the context of its limitations. First, this study was performed on clinical samples from a limited area of Japan. Further studies are needed to confirm the utility of this HRM-based assay using a larger size of samples from various regions. Second, the low-copy virus samples with a Ct value less than 35 may more frequently result in a false positive or false negative. The limit of detection should be determined using low-copy virus samples. Third, our assay cannot discriminate between Omicron variants BA.1 and BA.3 because these sub-lineages possess the same mutational spectra at R408, G446S/L452, and S477N/T478K (Table 1). Even if the variant BA.3 replaces BA.1 and BA.2, we can identify BA.3 by determining the D405N mutation in combination with others. In conclusion, we developed a novel assay to identify the main Omicron sub-lineages BA.1 and BA.2, using HRM analysis. As this HRM-based genotyping assay does not require sequence-specific probes, unlike the TaqMan probe assay, it is easy to perform and is cost-effective. Our results suggest that the current HRM-based assay is a powerful high-throughput tool for determining the SARS-CoV-2 Omicron sub-lineages. Since this assay was verified using limited clinical samples, further studies using diverse samples are needed to validate this HRM-based assay among the various institutions.

    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.

  2. Version published to 10.1101/2022.04.11.487970 on bioRxiv