Rapid And high throughput RT-qPCR assay for identification and differentiation between SARS-CoV-2 variants B.1.1.7 and B.1.351

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 activities and provide valuable data for epidemiological and policy decision making. We developed a multiplex quantitative RT-qPCR (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, and one that detects the 242-244 deletion, which is specific to variant B.1.351. The fourth reaction identifies 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 towards 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.