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Journal of Clinical Microbiology, November 2003, p. 5153-5158, Vol. 41, No. 11
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.11.5153-5158.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Microarrays for Genotyping Human Group A Rotavirus by Multiplex Capture and Type-Specific Primer Extension

Lovisa Lovmar,1 Caroline Fock,2 Felix Espinoza,3 Filemon Bucardo,3 Ann-Christine Syvänen,1 and Kåre Bondeson4*

Molecular Medicine,1 Clinical Bacteriology,2 Clinical Virology, Department of Medical Sciences, Uppsala University, 751 85 Uppsala, Sweden,4 Department of Microbiology, UNAN, León, Nicaragua3

Received 15 April 2003/ Returned for modification 3 June 2003/ Accepted 3 August 2003

Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses.

* Corresponding author. Mailing address: Department of Medical Sciences, Section of Virology, Uppsala University Hospital, 751 85 Uppsala, Sweden. Phone: 46-18 611 55 92. Fax: +46-18 51 56 15. E-mail: kare.bondeson{at}medsci.uu.se.

Journal of Clinical Microbiology, November 2003, p. 5153-5158, Vol. 41, No. 11
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.11.5153-5158.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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