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Development of a Microtiter Plate Hybridization-Based PCR-Enzyme-Linked Immunosorbent Assay for Identification of Clinically Relevant Human Group A Rotavirus G and P Genotypes

Instituto de Microbiologia, UFRJ, Rio de Janeiro, Brazil,¹ Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, Maryland,² Instituto Adolfo Lutz, São Paulo, Brazil,³ Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, Belém, Brazil,⁴ University of Tokyo, Tokyo, Japan,⁵ Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana,⁶ Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Coordinating Center for Infectious Diseases, CDC, Atlanta, Georgia⁷

Received 6 July 2007/ Returned for modification 31 August 2007/ Accepted 20 November 2007

A microtiter plate hybridization-based PCR-enzyme-linked immunosorbent assay (PCR-ELISA) has been used for the detection and identification of a variety of microorganisms. Here, we report the development of a PCR-ELISA for the identification of clinically relevant human rotavirus VP7 (G1 to G6, G8 to 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. 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 the United States. The results of this study showed that the PCR-ELISA was sensitive and easy to perform without the use of any expensive and sophisticated equipment, the reagents used are easy to obtain commercially and advantageous over multiplex PCR since more than one type-specific probe is used and the selection of probes is more flexible.

Published ahead of print on 5 December 2007.