Isothermal Point Mutation Detection: Toward a First-Pass Screening Strategy for Multidrug-Resistant Tuberculosis
Isothermal Point Mutation Detection: Toward a First-Pass Screening Strategy for Multidrug-Resistant Tuberculosis
复制标题
DOI：
10.1021/acs.analchem.7b01685
复制
发表时间：
2017-09-05
影响因子：
7.4
通讯作者：
Trau, Matt
中科院分区：
文献类型：
--
作者：
Ng, Benjamin Y. C.;Wee, Eugene J. H.;Trau, Matt
DNA 点突变是有用的生物标志物，可以提供关键的疾病分类，以实现准确诊断并为临床决策提供信息。检测点突变的传统方法通常基于实时聚合酶链式反应 (PCR) 或 DNA 测序等技术，这些技术通常速度较慢且需要昂贵的实验室设备。虽然已经提出了重组酶聚合酶扩增（RPA）等快速等温策略，但它们在区分点突变方面往往缺乏特异性。在此，我们描述了一种新策略，通过使用错配引物结合两轮富集过程，通过等温 DNA 扩增实现精确的点突变辨别。作为概念验证，该方法被应用于在特定条件下使用 RPA 快速、特异性地鉴定耐药结核分枝杆菌。该检测仅需要皮克水平的基因组 DNA 输入，其灵敏度和特异性足以检测 10% 的点突变负载，并且可以在 30 分钟内区分密切相关的突变体变体。该测定随后被应用于具有实时分析功能的低成本 3D 打印等温设备，以展示潜在的护理点应用。最后，通过检测其他三种临床上重要的癌症相关点突变，证明了该策略的普遍适用性。我们相信，我们的检测方法在广泛的医疗保健筛查过程中显示出潜力，可以在护理时对疾病表型进行检测和分类，从而减少这些情况下不必要的治疗和成本。
Point mutations in DNA are useful biomarkers that can provide critical classification of disease for accurate diagnosis and to inform clinical decisions. Conventional approaches to detect point mutations are usually based on technologies such as real-time polymerase chain reaction (PCR) or DNA sequencing, which are typically slow and require expensive lab-based equipment. While rapid isothermal strategies such as recombinase polymerase amplification (RPA) have been proposed, they tend to suffer from poor specificity in discriminating point mutations. Herein, we describe a novel strategy that enabled exquisite point mutation discrimination with isothermal DNA amplification, using mismatched primers in conjunction with a two-round enrichment process. As a proof of concept, the method was applied to the rapid and specific identification of drug-resistant Mycobacterium tuberculosis using RPA under specific conditions. The assay requires just picogram levels of genomic DNA input, is sensitive and specific enough to detect 10% point mutation loading, and can discriminate between closely related mutant variants within 30 min. The assay was subsequently adapted onto a low-cost 3D-printed isothermal device with real-time analysis capabilities to demonstrate a potential point-of-care application. Finally, the generic applicability of the strategy was shown by detecting three other clinically important cancer-associated point mutations. We believe that our assay shows potential in a broad range of healthcare screening processes for detecting and categorizing disease phenotypes at the point of care, thus reducing unnecessary therapy and cost in these contexts.