PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern

Abstract

With the emergence of SARS-CoV-2 variants that may increase transmissibility and/or cause escape from immune responses ^1-3 , there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant first detected in the UK ^4,5 could be serendipitously detected by the ThermoFisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern that lack spike Δ69-70, such as B.1.351 (also 501Y.V2) detected in South Africa ⁶ and P.1 (also 501Y.V3) recently detected in Brazil ⁷ . We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all three variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open source PCR assay to detect SARS-CoV-2 variants of concern ⁸ . Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence spread of B.1.1.7, B.1.351, and P.1.

Figure 1.. Identification of genome targets to differentiate between B.1.1.7, B. 1.351, P.1, and other SARS-CoV-2 lineages.

(a) Location on the SARS-CoV-2 genome where the targeted deletions in the ORF1a gene at amino acid positions 3675-3677 (Δ3675-3677) and the spike gene at amino acid positions 69-70 (Δ69-70) occur, (b) The Nextstrain ‘global build’ (nextstrain.org/ncov/global) accessed on 2021-01-22 showing the phylogenetic representation of 4,046 SARS-CoV-2 genomes colored by the presence of deletions at amino acid positions ORF1a 3575-3677 and spike 69-70. (c-f) Zooms of large SARS-CoV-2 clades, which include the variants of concern B.1.1.7, B. 1.351, and P.1, containing one or both deletions. A list of SARS-CoV-2 genomes used in the analysis is available in Source Data Fig. 1.

Figure 2.. ORF1a and spike target failure used to differentiate between SARS-CoV-2 variants of concern B.1.1.7, B. 1.351, P.1, and other lineages currently not of concern.

(a) Other lineages without target failure are detected by all three targets of the multiplex RT-qPCR assay, (b) Double target failure indicates the presence of the ORF1a Δ3675-3677 and spike Δ69-70 deletions and can be used to identify potential B.1.1.7 variants, (c) ORF1a target failure indicates the presence of the ORF1a Δ3675-3677 deletion, present in B.1.351 and P.1 variants of concern, (d) Spike target failure indicates presence of the spike Δ69-70 deletion, which is present in various lineages, including B.1.375. Shown are the Ct values for the N1, ORF1a, and spike primer-probe sets, with lines connecting Ct values obtained with the three sets for the same specimen. The dotted line indicates the limit of detection. VOC = variant of concern, VOI = variant of interest. Data used to make this figure can be found in Source Data Fig 2.