title: Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens.
abstract: 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 10(6) copies/mL, offering a 10-fold improvement over current rapid immunoassays.
authors.affiliation: †Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, United States.
authors.forename: Natalia M
authors.initials: NM
authors.lastname: Rodriguez
authors.affiliation: †Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, United States.
authors.forename: Jacqueline C
authors.initials: JC
authors.lastname: Linnes
authors.affiliation: †Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, United States.
authors.forename: Andy
authors.initials: A
authors.lastname: Fan
authors.affiliation: †Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, United States.
authors.forename: Courtney K
authors.initials: CK
authors.lastname: Ellenson
authors.affiliation: ‡Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, United States.
authors.forename: Nira R
authors.initials: NR
authors.lastname: Pollock
authors.affiliation: †Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, United States.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Klapperich
mesh_terms: D005821:Genetic Techniques / Q000191:economics / Q000295:instrumentation / Q000592:standards; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis*; D057230:Limit of Detection; D010209:Paper; D019095:Point-of-Care Systems; D012313:RNA / Q000737:chemistry / Q000235:genetics*
chemical_list: D012313:RNA