PCR-Free Methodology for Detection of Single-Nucleotide Polymorphism with a Cationic Polythiophene Reporter,ACS Sensors

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PCR-Free Methodology for Detection of Single-Nucleotide Polymorphism with a Cationic Polythiophene Reporter
ACS Sensors ( IF 8.2 ) Pub Date : 2021-02-23 , DOI: 10.1021/acssensors.0c02130
Muge Yucel ₁ , Altug Koc ₂ , Ayfer Ulgenalp ₂ , Gun Deniz Akkoc ₃ , Metin Ceyhan ₄ , Umit Hakan Yildiz _{3,

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Affiliation

This study presents a nonamplification-based nucleic acid assay for the detection of single-nucleotide polymorphism (SNP) associated with familial Mediterranean fever (FMF) besides polymerase chain reaction (PCR)-based methodologies. The major objective is to show the potential of the proposed assay for rapid screening of FMF in a Mediterranean region of 400 million population. The assay relies on binding difference of specially designed wild and mutant primers to the target genomic DNA, followed by determination of unbound primers by quick titration of a cationic polythiophene reporter. The fluorescent reporter exhibits signal transition from 525 to 580 nm in the presence of unbound primers, and it correlates the binding affinity of label-free primers to the homozygous wild and mutant genomes. As a proof of concept, 26 real samples are studied relying on the ON and OFF fluorescence signals of the cationic polythiophene reporter. The results are analyzed by principal component analysis (PCA), which provides clear separation of healthy and patient individuals. The further analysis by support vector machine (SVM) classification has revealed that our assay converges to 96% overall accuracy. These results support that the PCR-free nucleic acid assay has a significant potential for rapid and cost-effective screening of familial Mediterranean fever.

中文翻译：

用阳离子聚噻吩报告基因检测单核苷酸多态性的无 PCR 方法

本研究提出了一种基于非扩增的核酸检测方法，用于检测与家族性地中海热 (FMF) 相关的单核苷酸多态性 (SNP)，以及基于聚合酶链反应 (PCR) 的方法。主要目标是展示拟议的检测方法在 4 亿人口的地中海地区快速筛查 FMF 的潜力。该测定依赖于特殊设计的野生和突变引物与目标基因组 DNA 的结合差异，然后通过快速滴定阳离子聚噻吩报告基因来确定未结合的引物。在存在未结合的引物的情况下，荧光报告器表现出从 525 nm 到 580 nm 的信号转换，并将无标记引物与纯合野生和突变基因组的结合亲和力相关联。作为概念证明，根据阳离子聚噻吩报告分子的 ON 和 OFF 荧光信号研究了 26 个真实样品。结果通过主成分分析 (PCA) 进行分析，它提供了健康个体和患者个体的清晰分离。通过支持向量机 (SVM) 分类的进一步分析表明，我们的分析收敛到 96% 的总体准确度。这些结果支持无 PCR 核酸检测在快速和经济高效地筛查家族性地中海热方面具有巨大潜力。通过支持向量机 (SVM) 分类的进一步分析表明，我们的分析收敛到 96% 的总体准确度。这些结果支持无 PCR 核酸检测在快速和经济高效地筛查家族性地中海热方面具有巨大潜力。通过支持向量机 (SVM) 分类的进一步分析表明，我们的分析收敛到 96% 的总体准确度。这些结果支持无 PCR 核酸检测在快速和经济高效地筛查家族性地中海热方面具有巨大潜力。

更新日期：2021-03-26

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