A RT-qPCR system using a degenerate probe for specific identification and differentiation of SARS-CoV-2 Omicron (B.1.1.529) variants of concern

Abstract

Fast surveillance strategies are needed to control the spread of new emerging SARS-CoV-2 variants and gain time for evaluation of their pathogenic potential. This was essential for the Omicron variant (B.1.1.529) that replaced the Delta variant (B.1.617.2) and is currently the dominant SARS-CoV-2 variant circulating worldwide. RT-qPCR strategies complement whole genome sequencing, especially in resource lean countries, but mutations in the targeting primer and probe sequences of new emerging variants can lead to a failure of the existing RT-qPCRs. Here, we introduced an RT-qPCR platform for detecting the Delta- and the Omicron variant simultaneously using a degenerate probe targeting the key ΔH69/V70 mutation in the spike protein. By inclusion of the L452R mutation into the RT-qPCR platform, we could detect not only the Delta and the Omicron variants, but also the Omicron sub-lineages BA.1, BA.2 and BA.4/BA.5. The RT-qPCR platform was validated in small- and large-scale. It can easily be incorporated for continued monitoring of Omicron sub-lineages, and offers a fast adaption strategy of existing RT-qPCRs to detect new emerging SARS-CoV-2 variants using degenerate probes.

Fig 1. Detection of SARS-CoV-2 variants with the original ΔH69/V70 probe based on the SARS-CoV-2 alpha variant sequence.

(A) Detection of SARS-CoV-2 variants with the original probe, including patient samples confirmed as SARS-CoV-2 Alpha variant as positive control and SARS-CoV-2 negative patient samples and a water as negative controls. (B) Samples were tested as multiplex RT-qPCRs; E-Sarbeco in parallel with the ΔH69/V70 RT-qPCR. Error bars in (A-B) indicate SEM for three biological replicates.

Fig 2. Detection of SARS-CoV-2 variants with a degenerate ΔH69/V70 probe based on the SARS-CoV-2 BA.1 sequence.

(A) Detection of SARS-CoV-2 variants with a degenerate probe targeting the ΔH69/V70. The SARS-CoV-2 Alpha- and BA.1 variants were included as positive controls, and SARS-CoV-2 negative patient samples and a water as negative controls. (B) Detection of SARS-CoV-2 variants with the WT probe. SARS-CoV-2 WT, Delta and BA.2 variants were included as positive controls and SARS-CoV-2 negative patient samples and a water as negative controls.

Fig 3. Detection of ΔH69/V70 and L452R mutations in BA.1 and BA.2.

(A) Allelic discrimination analysis to differentiate between the H69/V70 deletion and the WT sequence. (B) Allelic discrimination analysis to differentiate between the 452R mutation and the L452 WT sequence.

Fig 4. Detection of ΔH69/V70 and L452R mutations in BA.4 and BA.5.

(A) Allelic discrimination analysis to differentiate between the ΔH69/V70 mutation and the WT sequence. (B) Allelic discrimination analysis to differentiate between the 452R mutation and the L452 WT sequence.