Multiplex PCR Assays for Identifying All Major SARS-CoV-2 Variants

Abstract

AbstractVariants of Concern (VOC) of SARS-CoV-2, including Alpha, Beta, Gamma, Delta, and Omicron threaten to prolong the pandemic leading to more global morbidity and mortality. Genome sequencing is the mainstay of tracking the development and evolution of the virus, but is costly, slow, and not easily accessible. A multiplex qRT-PCR assay for SARS-CoV-2 was developed, which identifies all VOC as well as other mutations of interest in the viral genome, eight mutations total, using single nucleotide discriminating molecular beacons in a two-tube assay. The presented variant molecular beacon assay showed a limit of detection of five copies of the viral RNA, with 100% specificity. Twenty-six SARS-CoV-2 positive patient samples were blinded and tested using this assay. When testing patient samples, the assay was in full agreement with results from deep sequencing with a sensitivity and specificity of 100% (26/26). We have used our design methodology to rapidly design an assay which detects the new Omicron variant. This Omicron assay was used to accurately identify this variant in 17 of 33 additional patient samples. These qRT-PCR assays identify all currently circulating VOC of SARS-CoV-2 as well as other important mutations in its Spike protein coding sequence. These assays can be easily implemented on broadly available five-color thermal cyclers and will help track the spread of these variants.