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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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posted on 04.08.2015, 00:00 by Natalia M. Rodriguez, Jacqueline C. Linnes, Andy Fan, Courtney K. Ellenson, Nira R. Pollock, Catherine M. Klapperich
The 2009 Influenza A (H1N1) pandemic disproportionately affected the developing world and highlighted 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.

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