Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection
| Autor: | Lázaro-Zaragozá, Ana, Maquieira Catala, Angel, Tortajada-Genaro, Luis Antonio |
|---|---|
| Rok vydání: | 2022 |
| Předmět: |
Fluid Flow and Transfer Processes
DNA biosensing Nucleotides Process Chemistry and Technology Nucleic Acid Hybridization Reproducibility of Results Hybridization chain reaction Bioengineering Allele-specific probe Cancer biomarker genes DNA biosensing allele-specific probe cancer biomarker genes hybridization chain reaction single-nucleotide mutation Single-nucleotide mutation QUIMICA ANALITICA Humans 03.- Garantizar una vida saludable y promover el bienestar para todos y todas en todas las edades Smartphone Instrumentation Alleles |
| Zdroj: | RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia instname ACS Sensors r-IIS La Fe. Repositorio Institucional de Producción Científica del Instituto de Investigación Sanitaria La Fe |
| ISSN: | 2379-3694 |
| DOI: | 10.1021/acssensors.1c02220 |
| Popis: | [EN] Massive DNA testing requires novel technologies to support a sustainable health system. In recent years, DNA superstructures have emerged as alternative probes and transducers. We, herein, report a multiplexed and highly sensitive approach 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 the HCR process on a chip to work with genomic DNA. The assay principle was demonstrated, and the variables for integrating the AS-HCR process and smartphone-based detection were also studied. The results were compared to a conventional polymerase reaction chain (PCR)-based test. The developed multiplex method enabled higher selectivity against single-base mismatch sequences at concentrations as low as 103 copies with a limit of detection of 0.7% of the mutant DNA percentage and good reproducibility (relative error: 5% for intra-assay and 17% for interassay). As proof of concept, 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 in KRAS and NRAS genes was validated, considering those obtained from the reference sequencing method. To conclude, AS-HCR is a rapid, simple, accurate, and cost-effective isothermal method that detects clinically relevant genetic variants and has a high potential for point-of-care demands. The authors acknowledge the financial support received from EU FEDER, the Spanish Ministry of Economy and Competitiveness (PID2019-110713RB-I00), and the Generalitat Valenciana (PROMETEO/2020/094 and GVA-FPI-2017 Ph.D. grant). |
| Databáze: | OpenAIRE |
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