Rapid on-site SNP discrimination across diverse mutational contexts

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Rapid identification of single-nucleotide polymorphism (SNP) mutations is essential for controlling infectious diseases and guiding treatment decisions.
• Existing diagnostic tools often require specialized equipment and time-consuming processes, limiting field applicability.

Purpose of the Study:

• To develop a novel, rapid, and field-deployable diagnostic technology for discriminating SNP-level mutations in double-stranded DNA (dsDNA).
• To assess the performance and robustness of this technology, including its ability to handle peripheral mutations.

Main Methods:

• A probe ligation-based assay was designed, where probes contain promoter and reporter sequences.
• Ligation triggers the generation of transcripts, enabling SNP detection.
• Results are visualized using lateral flow assay (LFA) strips, requiring no specialized equipment.

Main Results:

• The assay successfully discriminates SNP-level mutations within 20 minutes, even with pre-amplification.
• The technology demonstrated minimal influence from surrounding mismatches, allowing accurate mutation detection in complex sequences.
• Performance was validated using clinical samples of SARS-CoV-2 variants, confirming its effectiveness and mismatch endurance.

Conclusions:

• This ligation-based SNP detection technology offers a rapid, simple, and field-deployable solution for genetic analysis.
• Its straightforward probe design and LFA strip visualization make it accessible for diverse applications, including infectious disease diagnostics.
• The validated performance with clinical samples highlights its potential for real-world use in identifying genetic variations like those in viral variants.