Multiplex assay for simultaneously typing and subtyping influenza viruses by use of an electronic microarray.
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.
Huang
Ying
Y
Nanogen Inc, San Diego, CA 92121. yhuang@nanogen.com
Tang
Huong
H
Duffy
Stuart
S
Hong
Yuwen
Y
Norman
Sylvia
S
Ghosh
Madhu
M
He
Jie
J
Bose
Michael
M
Henrickson
Kelly J
KJ
Fan
Jiang
J
Kraft
Andrea J
AJ
Weisburg
William G
WG
Mather
Elizabeth L
EL
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
D012367:RNA, Viral; D014764:Viral Proteins