SARS-CoV-2 Variant Surveillance Using Tandem Targeted RT-PCR-based Genotyping Assays and Whole Genome Sequencing

Genomic surveillance is critical for tracking SARS-CoV-2 Variants of Concern (VOC) and for rapid detection of emerging variants. Whole genome sequencing (WGS) is the predominant method for genomic surveillance; but it is a laborious process for large-scale testing. The aim of this study was to assess the performance of a PCR-based mutation panel for the discrimination of 5 known VOC; Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529). Genotyping analysis was performed on 128 SARS-CoV-2 positive samples collected at the Life Science Testing Center at Northeastern University from April-December 2021. RNA extraction was performed using MagMax Viral/Pathogen II Nucleic Acid Isolation Kit. SARS-CoV-2 detection was confirmed using the TaqPath COVID-19 Combo Kit. Variant determination was conducted using a panel of TaqMan SARS-CoV-2 single nucleotide polymorphism (SNP) assays. On November 25, 2021, the emerging VOC (Omicron) was reported by South Africa and the panel was quickly modified to detect Omicron by substituting P681H and K417N assays. Based on the SNP panel analysis, variant identification in 128 samples were as follows: Alpha (N=34), Beta (N=1), Gamma (N=7), Delta (N=41) and Omicron (N=21). The genotyping panel accurately assigned lineages to 104 samples, confirmed by Ion Torrent GeneStudio S5 WGS. VOC discrimination using RT-PCR genotyping is a rapid, versatile method for detecting known and emerging SARS-CoV-2 variants. The versatility of SNP panels allows monitoring of emerging strains by simple layout adaptations. RT-PCR genotyping assays can expedite variant identification, enable high-throughput variant surveillance, and support WGS prioritization for detection of new variants.