Rapid on-site SNP discrimination across diverse mutational contexts

A diagnostic tool that can rapidly identify mutations at the single-nucleotide polymorphism (SNP) level in the field is crucial to control infectious diseases and select appropriate treatments. Here, we report a technology that can distinguish SNPs in dsDNA samples, based on the ligation of probes. Each probe contains the promoter and reporter sequence so that the transcripts, including the reporter sequence, can be generated when ligation occurs. This assay can discriminate SNP-level mutations within 20 min, followed by pre-amplification. Moreover, the minimal influence from surrounding mismatches permitted the discrimination of the mutation even in the presence of peripheral mutations in the target sequence. The diagnosis does not require specialized equipment, and results can be visualized with LFA strips. The straightforward design rule of the probes obviates the necessity for a time-consuming design process and empirical testing. The SNP discrimination performance and mismatch endurance of the technology were also validated using the clinical samples of SARS-CoV-2 variants.