Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens.
Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens.
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DOI：
10.1021/acs.analchem.5b01594
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发表时间：
2015-08-04
影响因子：
7.4
通讯作者：
Klapperich CM
中科院分区：
文献类型：
--
作者：
Rodriguez NM;Linnes JC;Fan A;Ellenson CK;Pollock NR;Klapperich CM
2009年甲型流感（H1N1）大流行对发展中国家的影响尤为严重，突出表明了传统诊断方法的主要不足之处，使其不适合在资源有限的环境中使用，从昂贵的设备和基础设施要求到令人无法接受的漫长周转时间。虽然快速免疫测定诊断测试成本低得多，更适合于具体情况，但它们的灵敏度极低，假阴性率高。在这些发展中地区，需要一种快速、低成本和独立于实验室基础设施的准确、灵敏和特异的分子诊断，以进行有效的即时检测和流行病学控制。我们开发了一种基于纸张的检测方法，可以通过聚醚砜纸基质直接从人临床鼻咽标本中提取和纯化RNA，直接在同一纸基质内进行H1N1特异性原位等温扩增，并在侧向流条上立即进行目视检测。完整的样本到答案检测可以在45分钟内在护理点进行，而不需要昂贵的设备或实验室基础设施，并且它具有106拷贝/mL的临床相关病毒载量检测限，比目前的快速免疫检测提高了10倍。
The 2009 Influenza A (H1N1) pandemic disproportionately affected the developing world and high-lighted the key inadequacies of traditional diagnostic methods that make them unsuitable for use in resource-limited settings, from expensive equipment and infrastructure requirements to unacceptably long turnaround times. While rapid immunoassay diagnostic tests were much less costly and more context-appropriate, they suffered from drastically low sensitivities and high false negative rates. An accurate, sensitive, and specific molecular diagnostic that is also rapid, low-cost, and independent of laboratory infrastructure is needed for effective point-of-care detection and epidemiological control in these developing regions. We developed a paper-based assay that allows for the extraction and purification of RNA directly from human clinical nasopharyngeal specimens through a poly(ether sulfone) paper matrix, H1N1-specific in situ isothermal amplification directly within the same paper matrix, and immediate visual detection on lateral flow strips. The complete sample-to-answer assay can be performed at the point-of-care in just 45 min, without the need for expensive equipment or laboratory infrastructure, and it has a clinically relevant viral load detection limit of 106 copies/mL, offering a 10-fold improvement over current rapid immunoassays.