Genotyping SARS-CoV-2 Variants Using Ratiometric Nucleic Acid Barcode Panels

Designing diagnostic assays to genotype rapidly mutating viruses remains a challenge despite the overall improvements in nucleic acid detection technologies. RT-PCR and next-generation sequencing are unsuitable for genotyping during outbreaks or in point-of-care detection due to their infrastructure requirements and longer turnaround times. We developed a quantum dot barcode multiplexing system to genotype mutated viruses. We designed multiple quantum dot barcodes to target conserved, wildtype, and mutated regions of SARS-CoV-2. We calculated ratios of the signal output from different barcodes that enabled SARS-CoV-2 detection and identified SARS-CoV-2 variant strains from a sample. We detected different sequence types, including conserved genes, nucleotide deletions, and single nucleotide substitutions. Our system detected SARS-CoV-2 patient specimens with 98% sensitivity and 94% specificity across 91 patient samples. Further, we leveraged our barcoding and ratio system to track the emergence of the N501Y SARS-CoV-2 mutation from December 2020 to May 2021 and demonstrated that the more transmissible N501Y mutation started to dominate infections by April 2021. Our barcoding and signal ratio approach can genotype viruses and track the emergence of viral mutations in a single diagnostic test. This technology can be extended to tracking other viruses. Combined with smartphone detection technologies, this assay can be adapted for point-of-care tracking of viral mutations in real time.

Automated summary

Designing diagnostic assays for rapidly mutating viruses is challenging, but we developed a quantum dot barcode multiplexing system that can genotype mutated viruses. By designing multiple quantum dot barcodes, we were able to detect and identify different sequence types of SARS-CoV-2, including conserved genes, nucleotide deletions, and single nucleotide substitutions. Our system showed high sensitivity and specificity in detecting SARS-CoV-2 patient specimens and tracking the emergence of viral mutations over time. This technology, combined with smartphone detection, has the potential for real-time point-of-care tracking of viral mutations.