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Journal of Clinical Microbiology, August 2007, p. 2641-2648, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00736-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development and Validation of DNA Microarray for Genotyping Group A Rotavirus VP4 (P[4], P[6], P[8], P[9], and P[14]) and VP7 (G1 to G6, G8 to G10, and G12) Genes{triangledown},{dagger}

Shinjiro Honma,1,{ddagger} Vladimir Chizhikov,2 Norma Santos,1,3 Masatoshi Tatsumi,1 Maria do Carmo S. T. Timenetsky,4 Alexandre C. Linhares,5 Joana D'Arc P. Mascarenhas,5 Hiroshi Ushijima,6 George E. Armah,7 Jon R. Gentsch,8 and Yasutaka Hoshino1*

Epidemiology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland,1 Laboratory of Method Development, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland,2 Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janerio, Brazil,3 Instituto Adolfo Lutz, São Paulo, Brazil,4 Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, Belém, Brazil,5 University of Tokyo, Tokyo, Japan,6 University of Ghana, Legon, Ghana,7 Gastroenteritis and Respiratory Viruses Laboratory Branch, Centers for Disease Control and Prevention, Atlanta, Georgia8

Received 4 April 2007/ Returned for modification 14 May 2007/ Accepted 4 June 2007

Previously, we reported the development of a microarray-based method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) (V. Chizhikov et al., J. Clin. Microbiol. 40:2398-2407, 2002). The expanded version of the rotavirus microarray assay presented herein is capable of identifying (i) five clinically relevant human rotavirus VP4 genotypes (P[4], P[6], P[8], P[9], and P[14]) and (ii) five additional human rotavirus VP7 genotypes (G5, G6, G8, G10, and G12) on one chip. Initially, a total of 80 cell culture-adapted human and animal reference rotavirus strains of known P (P[1] to P[12], P[14], P[16], and P[20]) and G (G1-6, G8 to G12, and G14) genotypes isolated in various parts of the world were employed to evaluate the new microarray assay. All rotavirus strains bearing P[4], P[6], P[8], P[9], or P[14] and/or G1 to G6, G8 to G10, or G12 specificity were identified correctly. In addition, cross-reactivity to viruses of genotype G11, G13, or G14 or P[1] to P[3], P[5], P[7], P[10] to P[12], P[16], or P[20] was not observed. Next, we analyzed a total of 128 rotavirus-positive human stool samples collected in three countries (Brazil, Ghana, and the United States) by this assay and validated its usefulness. The results of this study showed that the assay was sensitive and specific and capable of unambiguously discriminating mixed rotavirus infections from nonspecific cross-reactivity; the inability to discriminate mixed infections from nonspecific cross-reactivity is one of the inherent shortcomings of traditional multiplex reverse transcription-PCR genotyping. Moreover, because the hybridization patterns exhibited by rotavirus strains of different genotypes can vary, this method may be ideal for analyzing the genetic polymorphisms of the VP7 or VP4 genes of rotaviruses.

* Corresponding author. Mailing address: National Institutes of Health, Building 50, Room 6308, 50 South Drive, MSC 8026, Bethesda, MD 20892-8026. Phone: (301) 594-1851. Fax: (301) 480-1387. E-mail: thoshino{at}niaid.nih.gov

{triangledown} Published ahead of print on 13 June 2007.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

{ddagger} Present address: Department of Pediatrics, Sapporo University School of Medicine, Sapporo, Japan.

Journal of Clinical Microbiology, August 2007, p. 2641-2648, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00736-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Miller, M. B., Tang, Y.-W. (2009). Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology. Clin. Microbiol. Rev. 22: 611-633 [Abstract] [Full Text]  


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