Duplex RTqPCRs for detection and relative quantification of SARS-CoV-2 variants of concern (VOC)

During the evolution of the SARS-CoV-2 pandemic, new variants of the virus have emerged and spread worldwide. The increased transmissibility and proclivity of some of these variants to cause more serious disease threatens public health responses against the virus, thus being its monitoring relevant. While Next-Generation-Sequencing (NGS) is the gold standard to identify variants, it cannot always be rapidly implemented in some settings to provide information as an early warning tool. Duplex quantitative real time RTqPCR assays offer a sensitive and easy-to-use tool to detect, discriminate, and estimate relative proportions of SARS-CoV-2 variants containing signature mutations from variants lacking it, using allelic discrimination probes. We developed ten multiplexed RTqPCR assays that can detect Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron (B.1.1.529), Omicron BA.1 (B.1.1.529.1), Omicron BA.2 (B.1.1.529.2), Omicron FLip (XBB.1.5-like + L455F + F456L), Omicron BA.2.86 (B.1.1.529.2.86) and Omicron DeFLiRT (JN.1 + delS31) variants by targeting 21765_21770DelTACATG, 22281_22289DelCTTTACTTG, 28262_28263InsAACA, 22029_22034DelAGTTCA, 28362_28370DelGAGAACGCA, 22121InsGAGCCAGAA, 21633_21641DelTACCCCCTG, G22927T, T22928C and T22930A (L455F and F456L SNVs), ins21610ATGCCGCTGTTT and 21654_21656DelCTT deletions, insertions and SNVs in their genomes. Alpha, Beta, Delta, Omicron BA.1, Omicron BA.2, Omicron Flip, Omicron BA.2.86 and Omicron DeFLiRT markers are mapped to the S gene (residues 69/70, 241/243, 157/158, 214, 25/27, 455/456, 16 and 31 respectively), Gamma insertion is located between the end of ORF8 and the beginning of N gene, and Omicron (B.1.1.529) deletion is mapped to the N gene (residues 31/33). The duplex RTqPCR assay targeting 21765-21770DelTACATG mutation affecting residues HV69/70 has been previously used to estimate the relative proportion of Alpha VOC (Carcereny et al., 2021), and is also suitable to estimate the proportions of Omicron BA.4 and BA.5. All duplex RTqPCR assays targeting signature mutations may be used as a complementary tool to NGS for rapid variant tracking and surveillance in wastewater-based epidemiology.