Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection

Massive DNA testing requires novel technologies tosupport a sustainable health system. In recent years, DNAsuperstructures have emerged as alternative probes and trans-ducers. We, herein, report a multiplexed and highly sensitiveapproach based on an allele-specific hybridization chain reaction(AS-HCR) in the array format to detect single-nucleotide variants.Fast isothermal amplification was developed before activating theHCR process on a chip to work with genomic DNA. The assayprinciple was demonstrated, and the variables for integrating theAS-HCR process and smartphone-based detection were alsostudied. The results were compared to a conventional polymerasereaction chain (PCR)-based test. The developed multiplex methodenabled higher selectivity against single-base mismatch sequences at concentrations as low as 103copies with a limit of detection of0.7% of the mutant DNA percentage and good reproducibility (relative error: 5% for intra-assay and 17% for interassay). As proof ofconcept, the AS-HCR method was applied to clinical samples, including human cell cultures and biopsied tissues of cancer patients.Accurate identification of single-nucleotide mutations inKRASandNRASgenes was validated, considering those obtained from thereference sequencing method. To conclude, AS-HCR is a rapid, simple, accurate, and cost-effective isothermal method that detectsclinically relevant genetic variants and has a high potential for point-of-care demands.

Automated summary

This study presents a multiplexed and highly sensitive approach based on allele-specific hybridization chain reaction (AS-HCR) for detecting single-nucleotide variants. The developed method is rapid, simple, accurate, and cost-effective, making it suitable for detecting clinically relevant genetic variants.