Digital PCR discriminates between SARS-CoV-2 Omicron variants and immune escape mutations

As SARS-CoV-2 continues to evolve, mutations arise that will allow the virus to evade immune defenses and therapeutics. Assays that can identify these mutations can be used to guide personalized patient treatment plans. Digital PCR (dPCR) is a fast and reliable complement to whole genome sequencing that can be used to discriminate single nucleotide polymorphisms (SNPs) in template molecules. Here, we developed a panel of SARS-CoV-2 dPCR assays and demonstrate its applications for typing variant lineages and therapeutic monoclonal antibody resistance. We designed multiplexed dPCR assays for SNPs located at residue 3395 in the orf1ab gene and residue 143 of the spike gene that differentiate the Delta, Omicron BA.1, and Omicron BA.2 lineages. We demonstrate their validity on 596 clinical saliva specimens that were sequence-verified using Illumina whole genome sequencing. Next, we developed dPCR assays for spike mutations R346T, K444T, N460K, F486V, and F486S mutations that are associated with host immune evasion and reduced therapeutic monoclonal antibody efficacy. We demonstrate that these assays can be run individually or multiplexed to detect the presence of up to 4 SNPs in a single assay. We validate these dPCR assays on 81 clinical saliva SARS-CoV-2 positive specimens from Omicron subvariants BA.2.75.2, BM.1.1, BN.1, BF.7, BQ.1, BQ.1.1, and XBB. Thus, dPCR could serve as a useful tool to determine if clinical specimens contain therapeutically relevant mutations and inform patient treatment.