Detection and subtyping of influenza A virus based on a short oligonucleotide microarray

Diagn Microbiol Infect Dis. 2009 Nov;65(3):261-70. doi: 10.1016/j.diagmicrobio.2009.07.016. Epub 2009 Sep 5.

Abstract

We report the design and characterization of a microarray with 46 short virus-specific oligonucleotides for detecting influenza A virus of 5 subtypes: H1N1, H1N2, H3N2, H5N1, and H9N2. A unique combination of 3 specific modifications was introduced into the microarray assay: (1) short probes of 19 to 27 nucleotides, (2) simple amplification of full-length hemagglutinin and neuraminidase cDNAs with universal primers, and (3) Klenow-mediated labeling and further amplification of the samples before hybridization. The assay correctly and specifically detected and subtyped 11 different influenza A isolates from human, avian, and swine species representing the 5 subtypes. When tested with 225 clinical samples, 20 were detected to be positive using our microarray-based assay, whereas only 10 were positive by the conventional culture method. The entire analysis was completed within 7 h. Thus, these modifications result in a specific, sensitive, and rapid microarray assay and may be used for constructing microarrays for the detection of all influenza subtypes and strains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescence
  • Humans
  • Influenza A Virus, H1N1 Subtype / classification
  • Influenza A Virus, H1N1 Subtype / genetics
  • Influenza A Virus, H1N2 Subtype / classification
  • Influenza A Virus, H1N2 Subtype / genetics
  • Influenza A Virus, H3N2 Subtype / classification
  • Influenza A Virus, H3N2 Subtype / genetics
  • Influenza A Virus, H5N1 Subtype / classification
  • Influenza A Virus, H5N1 Subtype / genetics
  • Influenza A virus / classification*
  • Influenza A virus / genetics
  • Influenza, Human / diagnosis
  • Influenza, Human / virology*
  • Oligonucleotide Array Sequence Analysis / methods*
  • Oligonucleotide Probes
  • RNA, Viral / analysis
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Sensitivity and Specificity

Substances

  • Oligonucleotide Probes
  • RNA, Viral