An Isothermal Amplification Method for SARS-CoV-2 Variant Differentiation via Targeted Genomic RNA Detection

The rapid emergence of SARS-CoV-2 variants underscores the need for accurate, rapid, and affordable diagnostic tools, particularly in resource-limited settings. An isothermal amplification-based assay was developed integrating reverse-transcriptase recombinase polymerase amplification (RT-RPA), T7 transcription, and duplex-specific nuclease (DSN)-mediated detection for variant discrimination. The assay targets three genomic regions: a conserved region within ORF1a and two variant regions, ORF1a (Δ3675–3677) and the S gene (Δ69–70), enabling differentiation between the Wuhan-Hu-1 reference isolate and the B.1.1.7 variant. The method demonstrated high specificity and a limit of detection of 200 copies per sample using low-cost instrumentation. DSN-mediated cleavage improved discrimination between matched and mismatched RNA targets while enabling signal amplification through target recycling. The assay requires minimal laboratory infrastructure, relying on a heat block and fluorescent plate reader. These results demonstrate a scalable and cost-effective strategy for SARS-CoV-2 variant screening with potential as a future strategy for pathogen screening and variant surveillance.