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

Oran Erster, Ella Mendelson, Virginia Levy, Areej Kabat, Batya Mannasse, Hadar Asraf, Roberto Azar, Yaniv Ali, Rachel Shirazi, Efrat Bucris, Dana Bar-Ilan, Orna Mor, Michal Mandelboim, Danit Sofer, Shai Fleishon, Neta S. Zuckerman

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Emerging SARS-CoV-2 (SC-2) variants with increased infectivity and vaccine resistance are of major concern. Rapid identification of such variants is important for the public health decision making and to provide valuable data for epidemiological and policy decision making. We developed a multiplex reverse transcriptase quantitative PCR (RT-qPCR) assay that can specifically identify and differentiate between the emerging B.1.1.7 and B.1.351 SC-2 variants. In a single assay, we combined four reactions-one that detects SC-2 RNA independently of the strain, one that detects the D3L mutation, which is specific to variant B.1.1.7, one that detects the 242 to 244 deletion, which is specific to variant B.1.351, and the fourth reaction, which identifies the human RNAseP gene, serving as an endogenous control for RNA extraction integrity. We show that the strain-specific reactions target mutations that are strongly associated with the target variants and not with other major known variants. The assay's specificity was tested against a panel of respiratory pathogens (n = 16), showing high specificity toward SC-2 RNA. The assay's sensitivity was assessed using both in vitro transcribed RNA and clinical samples and was determined to be between 20 and 40 viral RNA copies per reaction. The assay performance was corroborated with Sanger and whole-genome sequencing, showing complete agreement with the sequencing results. The new assay is currently implemented in the routine diagnostic work at the Central Virology Laboratory, and may be used in other laboratories to facilitate the diagnosis of these major worldwide-circulating SC-2 variants.

Original languageEnglish
Article numbere00506-21
JournalMicrobiology spectrum
Volume9
Issue number2
DOIs
StatePublished - Oct 2021
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2021 American Society for Microbiology. All rights reserved.

Keywords

  • Alpha variant
  • Beta variant
  • Differential PCR
  • Illumina sequencing
  • Molecular diagnostics
  • Molecular virology
  • RT-qPCR
  • Rapid diagnosis
  • Real-time PCR
  • SARS-CoV-2
  • SC-2 variants
  • Sanger sequencing
  • Variant B.1.1.7
  • Variant B.1.351

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