A universal locked nucleic acid-integrated X-shaped DNA probe design for amplified fluorescence detection of single-nucleotide variant

Single-nucleotide variant (SNV) is crucial in clinical diagnostics as a useful biomarker, because it is closely associated with many diseases, such as genetic disorders and tumors. In this study, an enzyme-free and selective fluorescence biosensor is proposed for β-thalassemia SNV detection based on X-shaped probe, locked nucleic acid (LNA), and toehold-mediated strand-displacement reaction (TMSDR)-assisted recycling technology. On one hand, the LNA-integrated X-shaped probe has high recognition ability for SNV sequence even in the presence of a large amount of wild-type sequence, i.e., the rare SNV can be detected down to 0.01% variant allele frequency (VAF). On the other hand, the introduction of TMSDR-assisted recycling amplification system remarkably improves the sensitivity of the sensor with the detection limit as low as 6 fM mutation gene. Moreover, the designed X-shaped probe is universal because its functionalized oligonucleotides could be decoupled from the target-specific regions. These experimental results demonstrate that this developed method offers high specificity and sensitivity for SNV detection, which might be a potential alternative as an effective mutation detection technology for the detection of β-thalassemia SNV and other gene-related diseases.