Surveillance of SARS-CoV-2 lineage B.1.1.7 in Slovakia using a novel, multiplexed RT-qPCR assay

Abstract

The recent emergence of a novel variant of SARS-CoV-2 called lineage B.1.1.7 has sparked global alarm due to evidence of increased transmissibility, mortality, and uncertainty about vaccine efficacy, thus accelerating worldwide efforts to detect and track the variant. Current approaches to detect the B.1.1.7 variant include sequencing and molecular tests that contain a target assay that fails or results in reduced sensitivity towards the B.1.1.7 variant. Since many countries lack a robust genomic surveillance program and the failed target assays detect multiple unrelated variants that contain similar mutations as B.1.1.7, there is an urgent need to develop molecular tests that can accurately and rapidly identify the B.1.1.7 variant. We have developed a room temperature-stable, multiplexed RT-qPCR test that readily differentiates the B.1.1.7 variant from the most common SARS-CoV-2 variants. The test consists of two assays that target either the common SARS-CoV-2 spike gene or the two deletions in the spike gene (ΔH69/ΔV70 and ΔY144) that are present in the B.1.1.7 variant. Moreover, a simple relative comparison of the Ct values of the two assays permits not only identification of the B.1.1.7 variant but also its differentiation from other variants that harbor only the ΔH69/ΔV70 deletion. Each assay is multiplexed with a human RNase P internal control to assess RNA extraction and assay performance. This test can easily be implemented in diagnostic labs to rapidly scale B.1.1.7 surveillance efforts and is particularly useful in countries with high prevalence of variants possessing only the ΔH69/ΔV70 deletion because current strategies using target failure assays incorrectly identify these as putative B.1.1.7 variants.

Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection

“…Spike gene mutations such as N501Y, E484K/Q, L452R, and P681H/R have occurred independently in multiple VOCs and VOIs (variants of interest), enabling dynamic adaptation of existing assay designs to detect emerging SARS-CoV-2 variants [7]. RT-qPCR is the gold standard for detection of SARS-CoV-2 in clinical samples [8] and has also been used for SARS-CoV-2 spike gene mutation detection in previous studies, often focusing on the HV69/70 deletion and the N501Y SNP (single nucleotide polymorphism) using various methods such as specific primers, simple probes, and melt curve analyses [9][10][11][12][13][14][15]. Locked nucleic acid (LNA)-Probes represent a well described method for specifically detecting SNPs by RT-PCR [16][17][18].…”

A SARS-CoV-2 mutant from B.1.258 lineage with ∆H69/∆V70 deletion in the Spike protein circulating in Central Europe in the fall 2020

“…A newly developed RT-qPCR assay differentiating B.1.1.7 from other ∆H69/∆V70 variants [17] indicated that out of 122 clinical samples from a mass testing campaign in the city of Trenčín (Slovakia) in December 2020, only 5 (4.1%) belonged to the B.1.1.7 lineage, while an additional 50 (41.0%) samples carried the ∆H69/∆V70 deletion but were not B.1.1.7 (selected samples were later confirmed as B.1.258 ∆H69/∆V70 by sequencing). A routine RT-qPCR assay showed significantly lower Ct values in the swab samples for both B.1.1.7 and B.1.258 ∆H69/∆V70 samples, reflecting higher viral loads in patients carrying these variants (Fig.…”

“…Nextclade lineages [20] in color. Pangolin lineages [12] [17]. The real-time PCR was performed on a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems, Foster City, California, USA).…”

Multi-site Evaluation of SARS-CoV-2 Spike Mutation Detection Using a Multiplex Real-time RT-PCR Assay

“…Several different laboratory tests have been described [42-47], or are commercially available, which can help to identify specific mutations in the S gene of SARS-CoV-2. These tests are designed to detect certain VOC without the need for sequencing, and have varying sets of mutations targeted and other performance characteristics.…”