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.
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.
Departamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Cidade Universitária, CCS-Bl. I, Ilha do Fundão, Rio de Janeiro 21.941-590, Brazil. nsantos@micro.ufrj.br
Norma
N
Santos
Shinjiro
S
Honma
Maria do Carmo S T
Mdo C
Timenetsky
Alexandre C
AC
Linhares
Hiroshi
H
Ushijima
George E
GE
Armah
Jon R
JR
Gentsch
Yasutaka
Y
Hoshino
D000956:Antigens, Viral / Q000235:genetics; D001938:Brazil; D036022:Capsid Proteins / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005243:Feces / Q000821:virology; D005838:Genotype; D005869:Ghana; D006801:Humans; D007564:Japan; D016133:Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000175:diagnosis / Q000821:virology*; D012680:Sensitivity and Specificity; D014481:United States
D000956:Antigens, Viral; D036022:Capsid Proteins; C060339:VP4 protein, Rotavirus; C050779:VP7 protein, Rotavirus