Rapid and Accurate Identification of SARS-CoV-2 Omicron Variants Using Droplet Digital PCR (RT-ddPCR)

Abstract

Background Mutations in the receptor binding domain of the SARS-CoV-2 Spike protein are associated with increased transmission or substantial reductions in vaccine efficacy, including in the recently described Omicron variant. The changing frequencies of these mutations combined with their differing susceptibility to available therapies have posed significant problems for clinicians and public health professionals. Objective To develop an assay capable of rapidly and accurately identifying variants including Omicron in clinical specimens to enable case tracking and/or selection of appropriate clinical treatment. Study Design Using three duplex RT-ddPCR reactions targeting four amino acids, we tested 419 positive clinical specimens from February to December 2021 during a period of rapidly shifting variant prevalences and compared genotyping results to genome sequences for each sample, determining the sensitivity and specificity of the assay for each variant. Results Mutation determinations for 99.7% of detected samples agree with NGS data for those samples, and are accurate despite wide variation in RNA concentration and potential confounding factors like transport medium, presence of additional respiratory viruses, and additional mutations in primer and probe sequences. The assay accurately identified the first 15 Omicron variants in our laboratory including the first Omicron in Washington State and discriminated against S-gene dropout Delta specimen. Conclusion We describe an accurate, precise, and specific RT-ddPCR assay for variant detection that remains robust despite being designed prior the emergence of Delta and Omicron variants. The assay can quickly identify mutations in current and past SARS-CoV-2 variants, and can be adapted to future mutations.

Digital PCR Applications in the SARS-CoV-2/COVID-19 Era: a Roadmap for Future Outbreaks

“…Also, viral mutations pose a potential challenge to nucleic acid testing methods, leading to the occurrence of FNRs ( 140 ). For example, some assays have reported S gene dropout or S gene target failure (SGTF) in RT-qPCR and RT-dPCR assays targeting SARS-CoV-2’s S gene due to mutations caused by the Alpha and Omicron variants ( 139 , 141 – 144 ). Although specific mutations might compromise the sensitivity of the assays, it has been suggested that such assays may be used as a surrogate marker for the variants pending confirmation by sequencing ( 139 , 143 , 145 ).…”

“…Building on the same assay ( 144 ), Mills et al sought to further develop the assay to be capable of identifying key mutations in the spike region of SARS-CoV-2 and applied it to rapidly and accurately detect the recently emerged Omicron variant in clinical specimens ( 142 ). In this study, 390 clinical specimens were first screened using the assay, and the results matched 99% with whole-genome sequencing (WGS) results.…”

“…The results of the 15 Omicron samples were also confirmed by sequencing results. This study also recorded the first Omicron case in Washington State ( 142 ). Finally, during the development of CoV2-ID, a 5-plex RT-qPCR assay capable of detecting the D614G mutation, RT-dPCR was used to delineate the quantification and detection limits of the assay ( 140 ).…”

“…Taken together, these reports highlight the potential of RT-dPCR for validating RT-qPCR assays to detect mutations and also for developing novel RT-dPCR assays to detect and continuously monitor SARS-CoV-2 variants. Although sequencing is required to specifically identify a novel variant, existing dPCR assays could provide evidence that the mutation occurred in the region of the assay as observed with S gene target failure/dropout ( 142 , 144 ). In the case of a mutation in the assay region, the PCR efficiency is lower, resulting in a lower fluorescence amplitude of positive partitions that may be clearly visible in the partition/droplet readout.…”

Identification of Omicron-Delta Coinfections Using PCR-Based Genotyping

“…No clinical information was available for HMIX1 and HMIX2. Samples 5BG and 8LH were also tested by a separate PCR assay to detect S-gene target failure (ThermoFisher TaqPath assay), and a droplet digital (RT-dd)PCR assay targeting four different loci in the spike gene (417K, 452L, 484E, 501N) ( 3 , 4 ) (Table S1, Fig. 1B ).…”