多路复用
聚合酶链反应
克拉斯
多重聚合酶链反应
DNA
计算生物学
多重位移放大
分子信标
环介导等温扩增
桑格测序
分子生物学
生物
基因
DNA测序
寡核苷酸
遗传学
突变
DNA提取
作者
Ana Lázaro,Ángel Maquieira,Luis A. Tortajada‐Genaro
出处
期刊:ACS Sensors
[American Chemical Society]
日期:2022-02-21
卷期号:7 (3): 758-765
被引量:19
标识
DOI:10.1021/acssensors.1c02220
摘要
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.
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