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Journal of Clinical Microbiology, February 2001, p. 696-704, Vol. 39, No. 2
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.2.696-704.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Typing and Subtyping Influenza Virus Using DNA Microarrays and Multiplex Reverse Transcriptase PCR

Jiping Li,1,2 Shu Chen,2 and David H. Evans1,*

Department of Molecular Biology and Genetics1 and Laboratory Services Division,2 The University of Guelph, Guelph, Ontario N1G 2W1, Canada

Received 21 September 2000/Accepted 15 November 2000

A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding ~500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods.

* Corresponding author. Mailing address: Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Phone: (519) 824-4120, ext. 2575. Fax: (519) 837-2075. E-mail: dhevans{at}uoguelph.ca.

Journal of Clinical Microbiology, February 2001, p. 696-704, Vol. 39, No. 2
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.2.696-704.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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