Ligation-Based Assay for Variant-Typing Without Sequencing: Application to SARS-CoV-2 Variants of Concern

Background: COVID-19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing. Methods: A ligation-based typing assay was developed to detect know variants of SARS-CoV-2 by identifying the presence of characteristic single-nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real-time PCR instrumentation for assay performance. Findings: The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold-standard of viral genome sequencing. It achieved an average limit of detection of 7.4 x 104 genome copies per mL in contrived nasopharyngeal samples. The ligation-based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by sequence alignment of clinical samples. Interpretation: The assay demonstrates potential for robust variant-typing with performance comparable to next generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource-dependency and generalizability could expand access to variant classification information for pandemic surveillance. Funding Information: US National Institutes of Health R01 (AI157827) (JES and FRH) and Centers for Disease Control and Prevention (75D30121C10094) (JES and SRD) funded the study. DJN thanks the National Science Foundation Graduate Research Fellowship (1937963).