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Journal of Clinical Microbiology, February 2009, p. 390-396, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01807-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Multiplex Assay for Simultaneously Typing and Subtyping Influenza Viruses by Use of an Electronic Microarray {triangledown}

Ying Huang,1* Huong Tang,1 Stuart Duffy,1 Yuwen Hong,1 Sylvia Norman,1 Madhu Ghosh,1,{dagger} Jie He,2,3 Michael Bose,2,3 Kelly J. Henrickson,2,3 Jiang Fan,2,3 Andrea J. Kraft,2,3 William G. Weisburg,1 and Elizabeth L. Mather1

Nanogen, Inc., San Diego, California 92121,1 Department of Pediatrics,2 Midwest Respiratory Virus Program, Medical College of Wisconsin, Milwaukee, Wisconsin 532263

Received 18 September 2008/ Accepted 1 December 2008

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 102 to 103 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.

* Corresponding author. Mailing address: Nanogen Inc., 10398 Pacific Center Court, San Diego, CA 92121. Phone: (858) 410-4979. Fax: (858) 410-4650. E-mail: yhuang{at}nanogen.com

{triangledown} Published ahead of print on 10 December 2008.

{dagger} Present address: Vectrant Technologies, 2222 Michelson Dr., Irvine, CA 92612.

Journal of Clinical Microbiology, February 2009, p. 390-396, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01807-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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