A novel, multiplexed RT-qPCR assay to distinguish lineage B.1.1.7 from the remaining SARS-CoV-2 lineages

The emergence of a novel SARS-CoV-2 variant called lineage B.1.1.7 sparked global alarm due to evidence of increased transmissibility, mortality, and uncertainty about vaccine efficacy, thus accelerating efforts to detect and track the variant. Current approaches to detect lineage B.1.1.7 include sequencing and RT-qPCR tests containing a target assay that fails or results in reduced sensitivity towards the B.1.1.7 variant. Since many countries lack robust genomic surveillance programs and failed assays detect multiple unrelated variants containing similar mutations as B.1.1.7, we sought to develop an RT-qPCR test that can accurately and rapidly differentiate the B.1.1.7 variant from other SARS-CoV-2 variants. We used bioinformatics, allele-specific PCR, and judicious placement of LNA-modified nucleotides to develop a test that differentiates B.1.1.7 from other SARS-CoV-2 variants. We validated the test on 106 clinical samples with lineage status confirmed by sequencing. Our room temperature-stable, multiplexed RT-qPCR test consists of two assays that target either the common SARS-CoV-2 spike gene or spike gene deletions specific to lineage B.1.1.7. 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 {Delta}H69/{Delta}V70 deletion.The test showed 97% clinical sensitivity at detecting lineage B.1.1.7. This test can easily be implemented in labs to rapidly scale B.1.1.7 surveillance efforts and is particularly useful in countries with high prevalence of variants possessing only the {Delta}H69/{Delta}V70 deletion because current strategies using target failure assays incorrectly identify these as putative B.1.1.7 variants.