Confirming Multiplex Q-PCR Use in COVID-19 with Next Generation Sequencing: Strategies for Epidemiological Advantage

  1. Rob E. Carpenter
  2. Vaibhav Tamrakar
  3. Harendra Chahar
  4. Tyler Vine
  5. Rahul Sharma

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Abstract

Rapid classification and tracking of emerging SARS-CoV-2 variants are critical for understanding the transmission dynamics and developing strategies for interrupting the transmission chain. Next-Generation Sequencing (NGS) is an exceptional tool for whole-genome analysis and deciphering new mutations. The technique has been instrumental in identifying the Variants of Concern and tracking this pandemic. However, NGS remains expensive and time-consuming for large-scale monitoring of COVID-19. This study analyzed a total of 78 de-identified samples that screened positive for SARS-CoV-2 from two timeframes, August 2020 and July 2021. All 78 samples were classified into WHO lineages by whole genome sequencing then compared with two commercially available Q-PCR assays for spike protein mutation(s). The data showed good concordance with Q-PCR and NGS analysis for specific SARS-COV-2 lineages and characteristic mutations.

Deployment of Q-PCR testing to detect known SARS-COV-2 variants may be extremely beneficial. These assays are quick and cost-effective, thus can be implemented as an alternative to sequencing for screening known mutations of SARS-COV-2 for clinical and epidemiological interest. The findings support the great potential for Q-PCR to be an effective strategy offering several COVID-19 epidemiological advantages.

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  1. ScreenIT

    SciScore for 10.1101/2022.02.22.481485: (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

    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:
    Although the unavailability of the raw data (FASTQ files) from the 67 samples remains the limitation of the study, phylogenetic analysis of the 20 samples tested at Advanta Analytical Laboratories were clustered as expected; all the VOC and Non-VOC samples were grouped appropriately. Although NGS is the most reliable method for detecting mutations in SARS-CoV-2, the methodology is not practically applicable for large-scale surveillance, particularly in resource-limited settings. Factors like continuous validation studies, logistic challenges, database validity, cost-benefit analysis, and high technical expertise make the implementation of NGS in routine clinical settings difficult. Comparatively, Q-PCR—a gold standard for diagnosing SARS-CoV-2—is a method that can be extended for variant detection and monitoring in clinical settings. The novelty of this research is that it demonstrated that Q-PCR is as effective as NGS in detecting SARS-CoV-2 mutations. Two Q-PCR-based assays for the detection of SARS-CoV-2 mutagenic variants were tested and compared with NGS data. Both assays were able to detect L452R mutation with 100% (67/67; GT Molecular) and 94% (63/67; Thermo Fisher) accuracy when compared to NGS. While NGS is an essential tool for sequencing the entire genome and identification of new mutations, this study suggests Q-PCR can aptly serve as an easy to deploy, cost-effective, and time-sensitive solution for the detection of known mutations for mass surveillance. Likewise...

    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.

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

    About SciScore

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  2. Version published to 10.1101/2022.02.22.481485v1 on bioRxiv