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

Fast surveillance strategies are needed to control the spread of new emerging SARS-CoV-2 variants and gain time for evaluation of their pathological 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 {Delta}H69/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.

Rapid surveillance platforms for key SARS-CoV-2 mutations in Denmark

Multiple mutations in SARS-CoV-2 variants of concern (VOCs) may increase, transmission, disease severity, immune evasion and facilitate zoonotic or anthoprozoonotic infections. Four such mutations, {Delta}H69/V70, L452R, E484K and N501Y, occur in the SARS-CoV-2 spike glycoprotein in combinations that allow detection of the most important VOCs. Here we present two flexible RT-qPCR platforms for small-and large-scale screening to detect these mutations, and schemes for adapting the platforms for future mutations. The large-scale RT-qPCR platform, was validated by pair-wise matching of RT-qPCR results with WGS consensus genomes, showing high specificity and sensitivity. Detection of mutations using this platform served as an important interventive measure for the Danish public health system to delay the emergence of VOCs and to gain time for vaccine administration. Both platforms are valuable tools for WGS-lean laboratories, as well for complementing WGS to support rapid control of local transmission chains worldwide.