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J. Clin. Microbiol. doi:10.1128/JCM.01361-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Development of a Microtiter Plate Hybridization-based PCR-enzyme-linked Immunosorbent Assay (PCR-ELISA) for Identification of Clinically Relevant Human Group A Rotavirus G and P Genotypes

Instituto de Microbiologia/UFRJ, Brazil; Laboratory of Infectious Diseases, NIAID/NIH, USA; Instituto Adolfo Lutz, Brazil; Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, Brazil; University of Tokyo, Japan; Noguchi Memorial Institute for Medical Research, University of Ghana, Ghana; Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Coordinating Center for Infectious Diseases, CDC, Atlanta GA

^* To whom correspondence should be addressed. Email: nsantos{at}micro.ufrj.br or nsosantos@gmail.com.

	Abstract

A microtiter plate hybridization-based PCR-enzyme-linked immunosorbent assay or PCR-ELISA has been used for detection and identification of a variety of microorganisms. Here we report the development of a PCR-ELISA method for the identification of clinically relevant human rotavirus VP7 (G1-G6, G8-G10, and G12) and VP4 (P[4], P[6], P[8], P[9] and P[14]) genotypes. The G and P types of reference human and animal rotavirus strains for which specific probes were available were correctly identified by the PCR-ELISA assay. In addition, reference strains bearing G or P genotypes for which specific probes were unavailable such as G11, G14, P[3], P[10] and P[11] did not display any cross-reactivity to the probes. The usefulness of the assay was further evaluated by analyzing a total of 396 rotavirus-positive stool samples collected in four countries: Brazil, Ghana, Japan and USA. The results of this study showed that the PCR-ELISA was (i) sensitive, (ii) easy to perform without use of any expensive and sophisticated equipment, (iii) the reagents used are easy to obtain commercially, and (iv) advantageous over the multiplex PCR since (a) more than one type-specific probe is used and (b) the selection of probes is more flexible.