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J Clin Microbiol 2007 Aug;45(8):2641-8

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-G6, G8-G10 and G12) Genes.

Honma S, Chizhikov V, Santos N, Tatsumi M, Timenetsky MD, Linhares AC, Mascarenhas JD, Ushijima H, Armah GE, Gentsch JR, Hoshino Y

Hoshino Y (reprint author), NIAID, Epidemiol Sect, Infect Dis Lab, NIH, Bldg 50,Room 6308,50 S Dr,MSC 8026, Bethesda, MD 20892 USA NIAID, Epidemiol Sect, Infect Dis Lab, NIH, Bethesda, MD 20892 USA US FDA, Ctr Biol Evaluat & Res, Lab Method Dev, Rockville, MD USA Univ Fed Rio de Janeiro, Inst Microbiol, BR-21941 Rio De Janeiro, Brazil Ins Adolf Lutz, Sao Paulo, Brazil Secretaria Vigilancia Saude, Inst Evandro Chagas, Belem, Para Brazil Univ Tokyo, Tokyo, Japan Univ Ghana, Legon, Ghana Ctr Dis Control & Prevent, Gastroenteritis Resp Viruses Lab Branch, Atlanta, GA USA


Previously, we reported the development of a microarray-based method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) (Chizhikov et al., J. Clin. Microbiol. 2002;40:2398-2407). The expanded version of the rotavirus microarray presented herein is capable of identifying (i) five clinically relevant human rotavirus VP4 genotypes (P[4], (6), (8), (9) and (14) and (ii) five additional human rotavirus VP7 genotypes (G5, 6, 8, 10, and 12) on one chip. Initially, a total of 80 cell culture-adapted human and animal reference rotavirus strains of known P (P[1]-[12], (14), (16) and (20) and G (G1-6, 8-12 and 14) genotypes isolated in various parts of the world were employed to evaluate the new microarray assay. All rotavirus strains bearing P[4], (6), (8), (9) or (14) and/or G1-6, 8-10 or 12 specificity were identified correctly. In addition, cross-reactivity to viruses bearing G11, 13 or 14, or P[1]-[3], (5), (7), (10)-[12], (16) or (20) was not observed. Next, we analyzed a total of 128 rotavirus-positive human stool samples collected in 3 countries (Brazil, Ghana and USA) 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 non-specific cross-reactivity, the latter being one of the inherent shortcomings of a traditional multiplex RT-PCR genotyping. Moreover, because the hybridization pattern exhibited by each rotavirus strain in a given genotype can vary, this method may be ideal to analyze genetic polymorphism of the VP7 or VP4 gene of rotaviruses.

Category: Journal Article
PubMed ID: #17567783
Entry Created: 2011-10-04 Entry Last Modified: 2012-08-29