title: Multiplex assay for simultaneously typing and subtyping influenza viruses by use of an electronic microarray.
abstract: We report on the use of an electronic microarray to simultaneously type influenza A and B viruses and to distinguish influenza A virus subtypes H1N1 and H3N2 from the potentially pandemic avian virus subtype H5N1. The assay targets seven genes: the H1, H3, H5, N1, and N2 genes of influenza A virus; the matrix protein M1 gene of influenza A virus; and the nonstructural protein (NS) gene of influenza B virus. By combining a two-step reverse transcription-multiplex PCR with typing and subtyping on the electronic microarray, the assay achieved an analytical sensitivity of 10(2) to 10(3) copies of transcripts per reaction for each of the genes. The assay correctly typed and subtyped 15 different influenza virus isolates, including two influenza B virus, five A/H1N1, six A/H3N2, and two A/H5N1 isolates. In addition, the assay correctly identified 8 out of 10 diluted, archived avian influenza virus specimens with complete typing and subtyping information and 2 specimens with partial subtyping information. In a study of 146 human clinical specimens that had previously been shown to be positive for influenza virus or another respiratory virus, the assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The assay is a rapid, accurate, user-friendly method for simultaneously typing and subtyping influenza viruses.
authors.affiliation: Nanogen Inc, San Diego, CA 92121. yhuang@nanogen.com
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.forename: Huong
authors.initials: H
authors.lastname: Tang
authors.forename: Stuart
authors.initials: S
authors.lastname: Duffy
authors.forename: Yuwen
authors.initials: Y
authors.lastname: Hong
authors.forename: Sylvia
authors.initials: S
authors.lastname: Norman
authors.forename: Madhu
authors.initials: M
authors.lastname: Ghosh
authors.forename: Jie
authors.initials: J
authors.lastname: He
authors.forename: Michael
authors.initials: M
authors.lastname: Bose
authors.forename: Kelly J
authors.initials: KJ
authors.lastname: Henrickson
authors.forename: Jiang
authors.initials: J
authors.lastname: Fan
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Kraft
authors.forename: William G
authors.initials: WG
authors.lastname: Weisburg
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Mather
mesh_terms: D005838:Genotype; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D053122:Influenza A Virus, H3N2 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D009981:Influenza B virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D046228:Microarray Analysis / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins