A rapid point-of-care population-scale dipstick assay to identify and differentiate SARS-CoV-2 variants in COVID-19-positive patients

Abstract Delta and Omicron variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are remarkably contagious, and have been recognized as variants of concern (VOC). The acquisition of spontaneous substitutions or insertion–deletion mutations (indels) in the spike protein-encoding gene substantially increases the binding affinity of the receptor binding domain (RBD)-hACE2 complex and upsurges the transmission of both variants. In this study, we analyzed thousands of genome sequences representing 30 different SARS-CoV-2 variants and identified the Delta and Omicron variants specific nucleic acid signatures in the spike gene. Based on the variant-specific nucleic acid sequences, we synthesized different oligos and optimized a multiplex PCR (mPCR) assay that can identify and differentiate the Delta and Omicron variants of SARS-CoV-2. We further extended our work on this mPCR and translated it into a dipstick assay by adding a tag linker sequence to the 5’ end of the forward primer and biotin to the 3’ end of the oligos. Streptavidin-coated latex beads and the dipstick imprinted with a probe for the tag linker sequence in the test strips were used for the detection assay. Our dipstick-based assay, developed as a rapid point-of-care test for identifying and differentiating SARS-CoV-2 variants has the potential to be used in low-resource settings and scaled up to the population level.