title: Development and clinical testing of a simple, low-density gel element array for influenza identification, subtyping, and H275Y detection.
abstract: The objectives of this study were to develop a user-friendly, gel element microarray test for influenza virus detection, subtyping, and neuraminidase inhibitor resistance detection, assess the performance characteristics of the assay, and perform a clinical evaluation on retrospective nasopharyngeal swab specimens. A streamlined microarray workflow enabled a single user to run up to 24 tests in an 8h shift. The most sensitive components of the test were the primers and probes targeting the A/H1 pdm09 HA gene with an analytical limit of detection (LoD) <100 gene copies (gc) per reaction. LoDs for all targets in nasopharyngeal swab samples were ≤1000 gc, with the exception of one target in the seasonal A/H1N1 subtype. Seasonal H275Y variants were detectable in a mixed population when present at >5% with wild type virus, while the 2009 pandemic H1N1 H275Y variant was detectable at ≤1% in a mixture with pandemic wild type virus. Influenza typing and subtyping results concurred with those obtained with real-time RT-PCR assays on more than 97% of the samples tested. The results demonstrate that a large panel of single-plex, real-time RT-PCR tests can be translated to an easy-to-use, sensitive, and specific microarray test for potential diagnostic use.
authors: Akonni Biosystems, Inc., 400 Sagner Avenue, Suite 300, Frederick, MD 21701, United States. Electronic address: dchandler@akonni.com.
authors: Darrell P
authors: DP
authors: Chandler
authors: Laboratory of Viral Diseases, Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208, United States.
authors: Sara B
authors: SB
authors: Griesemer
authors: Akonni Biosystems, Inc., 400 Sagner Avenue, Suite 300, Frederick, MD 21701, United States.
authors: Christopher
authors: C
authors: Knickerbocker
authors: Akonni Biosystems, Inc., 400 Sagner Avenue, Suite 300, Frederick, MD 21701, United States.
authors: Julia B
authors: JB
authors: Golova
authors: Akonni Biosystems, Inc., 400 Sagner Avenue, Suite 300, Frederick, MD 21701, United States.
authors: Amine
authors: A
authors: Lambarqui
authors: Akonni Biosystems, Inc., 400 Sagner Avenue, Suite 300, Frederick, MD 21701, United States.
authors: Alexander N
authors: AN
authors: Perov
authors: Akonni Biosystems, Inc., 400 Sagner Avenue, Suite 300, Frederick, MD 21701, United States.
authors: Cynthia
authors: C
authors: Zimmerman
authors: Akonni Biosystems, Inc., 400 Sagner Avenue, Suite 300, Frederick, MD 21701, United States.
authors: Cory
authors: C
authors: Wiles
authors: Akonni Biosystems, Inc., 400 Sagner Avenue, Suite 300, Frederick, MD 21701, United States.
authors: George B
authors: GB
authors: Rudy
authors: Laboratory of Viral Diseases, Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208, United States.
authors: Kirsten
authors: K
authors: St George
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D024882:Drug Resistance, Viral; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D007753:Laboratories; D046228:Microarray Analysis / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D058889:Molecular Typing / Q000379:methods; D050505:Mutant Proteins / Q000235:genetics; D009305:Nasopharynx / Q000821:virology; D009439:Neuraminidase / Q000235:genetics*; D053139:Oseltamivir / Q000494:pharmacology; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D013997:Time Factors; D014764:Viral Proteins / Q000235:genetics*; D000078329:Workforce
chemical_list: D000998:Antiviral Agents; D050505:Mutant Proteins; D014764:Viral Proteins; D053139:Oseltamivir; C487630:NA protein, influenza A virus; D009439:Neuraminidase
keywords: Epidemiology; Microarray; Molecular diagnostic; Oseltamivir resistance; Surveillance