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.
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.
Summary Sensitive and accurate RT‐qPCR tests are the primary diagnostic tools to identify SARS‐CoV‐2‐infected patients. While many SARS‐CoV‐2 RT‐qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands‐on‐time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS‐CoV‐2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS‐CoV‐2 RT‐qPCR tests that are originally based on the protocol targeting regions of the RNA‐dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS‐CoV‐2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature‐stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction‐free version enables high sensitivity detection of SARS‐CoV‐2 in about an hour using minimally invasive, self‐collected gargle samples. These RT‐qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS‐CoV‐2 and the most common seasonal influenzas during the vaccination phase of the pandemic.
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