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

Abstract

The emergence of a novel SARS-CoV-2 B.1.1.7 variant sparked global alarm due to increased transmissibility, mortality, and uncertainty about vaccine efficacy, thus accelerating efforts to detect and track the variant. Current approaches to detect 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 genomic surveillance programs and failed assays detect unrelated variants containing similar mutations as B.1.1.7, we used allele-specific PCR, and judicious placement of LNA-modified nucleotides to develop an RT-qPCR test that accurately and rapidly 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 country-wide surveillance study of B.1.1.7 prevalence in Slovakia. Our multiplexed RT-qPCR test showed 97% clinical sensitivity and retesting 6,886 SARS-CoV-2 positive samples obtained during three campaigns performed within one month, revealed pervasive spread of B.1.1.7 with an average prevalence of 82%. 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.

Figure 1

Development and optimization of a general SARS-CoV-2 S gene primer/probe set for all SARS-CoV-2 variants. Schematic illustrates genomic organization of SARS-CoV-2 with an emphasis on the location of both SARS-CoV-2 S gene and B.1.1.7 primer/probe sets relative to the ΔH69/ΔV70 and ΔY144 deletions (red lines with ball points) observed in the spike gene of B.1.1.7 variants.

Figure 2

Analytical sensitivity and clinical validation of SARS-CoV-2 S gene and B.1.1.7 assays. (a) The limit of detection was determined for both SARS-CoV-2 S gene and B.1.1.7 assays by serial dilutions of isolated viral B.1.1.7 RNA. Data depict the mean and SD of eight replicates per each dilution. The dotted line at Ct 40 serves as a threshold after which amplification is considered invalid. (b) Overview of ΔCt values (= B.1.1.7 assay Ct—SARS-CoV-2 S gene assay Ct) for each sample in the clinical validation. Symbols represent the various SARS-CoV-2 lineages that were identified by sequencing. Closed symbols represent samples correctly identified by either the SARS-CoV-2 S gene or B.1.1.7 assays, whereas open symbols denote samples that did not meet the criterion established for variant identification. The shaded background shows ΔCt ranges that correspond with the criterion to report a sample as B.1.1.7 positive (pink), ΔH69/ΔV70 deletion positive (teal), and inconclusive (gray). ND, not detected. (c) Decision tree demonstrating the proper workflow, interpretation criterion, and actions to implement the SARS-CoV-2 S gene and B.1.1.7 assays in a testing regime to identify B.1.1.7 positive samples.

Figure 3

Tracking the prevalence of lineage B.1.1.7 in Slovakia. (a) Maps of the prevalence of lineage B.1.1.7 in the eight regions of Slovakia during the three screening rounds held on February 2nd, 2021, February 17th, 2021, and March 3rd, 2021. Regions in red have prevalence > 80%, regions in yellow have prevalence < 60%. (b) Maps of the prevalence of lineage B.1.1.7 in the 79 districts of Slovakia during the three screening rounds held on February 2nd, 2021, February 17th, 2021, and March 3rd, 2021. Regions in red have prevalence > 80%, regions in yellow have prevalence < 60%. (c) Trends in B.1.1.7 prevalence during the three screening rounds in each of the eight regions of Slovakia. (d) Observed trends in B.1.1.7 prevalence during the three screening rounds and expected prevalence given various 2-week spread factors (shown in parentheses) assuming the prevalence of February 2nd, 2021. The prevalence maps in sections (a,b) were generated using Datawrapper (https://www.datawrapper.de/) in version 1.25.0.