Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València-Universitat de València, Camino de Vera S/n, E46022, Valencia, Spain; Unidad Mixta UPV-La Fe, Nanomedicine and Sensors, IIS La Fe, Valencia, Spain.
Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València-Universitat de València, Camino de Vera S/n, E46022, Valencia, Spain; Unidad Mixta UPV-La Fe, Nanomedicine and Sensors, IIS La Fe, Valencia, Spain; Departamento de Química, Universitat Politècnica de València, Valencia, Spain.
Anal Chim Acta. 2019 Dec 27;1092:49-56. doi: 10.1016/j.aca.2019.10.006. Epub 2019 Oct 13.
The studied challenge is the specific detection of low-abundant genomic variants that differ by a single nucleotide from the wild type. The combination of blocked recombinase polymerase amplification (RPA) and selective capture by probes immobilised on magnetic-core particles integrated into a flow system is presented. The sensing principle was demonstrated as the effective concentration-detection of the specific generated products was achieved. The analytical performance of resulting assay was successfully compared to PCR-based methods or array formats, providing faster effective detection of the selective products. As proof of concept, the single-nucleotide substitutions of the KRAS gene at codon 12 were studied in chip with parallel microchambers and permanent magnets. The blocked RPA products (generated at 37 °C) from tumour biopsies (extracted DNA 4 ng) provided a specific fluorescent bead-line that depends on the present mutation. The results agree with those reported by next-generation sequencing and provide new opportunities for in vitro diagnostic and personalised medicine.
本研究的挑战是特异性检测低丰度基因组变异，这些变异与野生型相比仅单个核苷酸不同。本研究提出了一种结合阻断重组酶聚合酶扩增（RPA）和探针固定在磁性核粒子上的选择性捕获的方法，并将其集成到一个流系统中。该传感原理通过有效检测特异性生成产物的浓度得到了证明。与基于 PCR 的方法或阵列格式相比，该方法具有更快的有效检测选择性产物的性能。作为概念验证，在带有平行微室和永久磁铁的芯片上研究了 KRAS 基因密码子 12 处的单核苷酸替换。来自肿瘤活检的阻断 RPA 产物（在 37°C 下生成）提供了一条依赖于突变的特异性荧光珠线。这些结果与下一代测序报告的结果一致，并为体外诊断和个性化医疗提供了新的机会。
