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

Background The emergence of a novel SARS-CoV-2 variant of concern called B.1.1.7 lineage 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. Aim 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. Methods 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 and conducted a surveillance study of B.1.1.7 lineage prevalence in Slovakia. Results Our multiplexed RT-qPCR test showed 97% clinical sensitivity at detecting lineage B.1.1.7. The assay was used in a country-wide surveillance of B.1.1.7 lineage spread in Slovakia. Retesting nearly 7,000 SARS-CoV-2 positive samples obtained during three campaigns performed within a one month period, revealed pervasive spread of B.1.1.7 with an average prevalence of 82%. Conclusion Labs can easily implement this test to rapidly scale B.1.1.7 surveillance efforts and it 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.