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Journal of Clinical Microbiology, July 2002, p. 2398-2407, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2398-2407.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Detection and Genotyping of Human Group A Rotaviruses by Oligonucleotide Microarray Hybridization

V. Chizhikov,1* M. Wagner,2 A. Ivshina,1 Y. Hoshino,2 A. Z. Kapikian,2 and K. Chumakov1

Laboratory of Method Development, Center for Biologics Evaluation and Research, Food and Drug Administration, Kensington, Maryland 20895,1 Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 208922

Received 1 March 2002/ Returned for modification 5 April 2002/ Accepted 22 April 2002

A rapid and reliable method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) of human rotaviruses based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides immobilized on the surface of glass slides were selected to bind to the multiple target regions within the VP7 gene that are highly conserved among individual rotavirus genotypes. Rotavirus cDNA was amplified in a PCR with primers common to all group A rotaviruses. A second round of nested PCR amplification was performed in the presence of indodicarbocyanine-dCTP and another pair of degenerate primers also broadly specific for all genotypes. The use of one primer containing 5'-biotin allowed us to prepare fluorescently labeled single-stranded hybridization probe by binding of another strand to magnetic beads. The identification of rotavirus genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The specificity of oligonucleotide microchip hybridization was evaluated by testing 20 coded rotavirus isolates from different geographic areas for which genotypes were previously determined by conventional methods. Analysis of the coded specimens showed that this microarray-based method is capable of unambiguous identification of all rotavirus strains. Because of the presence of random mutations, each individual virus isolate produced a unique hybridization pattern capable of distinguishing different isolates of the same genotype and, therefore, subgenotype differentiation. This strain information indicates one of several advantages that microarray technology has over conventional PCR techniques.

* Corresponding author. Mailing address: Laboratory of Method Development, Center for Biologics Evaluation and Research, Food and Drug Administration, HFM-470, 1401 Rockville Pike, Rockville, MD 20852. Phone: (301) 827-2872. Fax: (301) 827-4622. E-mail: chizhikov{at}cber.fda.gov.

Journal of Clinical Microbiology, July 2002, p. 2398-2407, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2398-2407.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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