Intended Use
Identification of specific G and P Rotavirus genotypes.

Summary and explanation
Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR method for amplification of rotavirus RNA and a reverse hybridization assay to detect amplimers and identify specific G and P genotypes present in human stools.
Novel broad spectrum PCR primers were developed for both VP4 and VP7, permitting amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and VP7 strips contain universal probes for detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contained type-specific probes for P4, P6, P8, P9 and P10. The VP7 strip contained type-specific probes for G1, G2, G3, G4, G5, G6, G8 and G9. In addition, the assay permits specific identification of the rotavirus G1[P8] strain, used in the RotarixTM vaccine.
Both RT-PCR methods were confirmed to permit detection of a broad spectrum of genotypes, as tested by analysis of multiple reference strains. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections of different genotypes. Rotavirus genotypes, for which no type-specific probes were present on the strip, were adequately identified by the universal detection probes. The assay was formally validated by analysis of specificity, sensitivity, reproducibility, precision, accuracy and robustness. This validated assay could be useful for large scale epidemiological and clinical trials.

Principles of the procedure
The RHA Rotavirus VP4 / VP7 genotyping test is based on the reverse hybridization principle. A part of the genome G and P Rotavirus genotypes are amplified by PCR, and denatured biotinylated amplicons are hybridized with specific oligonucleotide probes, which are immobilized as parallel lines on membrane strips. After hybridization and stringent washing, streptavidin-conjugated alkaline phosphatase is added and bound to any biotinylated hybrid previously formed. Incubation with BCIP/NBT chromogen yields in a purple precipitate and the results can be visually interpreted.
The test has been validated for G and P genotyping of Rota viruses in stool samples.

Ordering information
REF: S-1016 RHA kit Rotavirus VP4 48 tests
REF: S-1017 RHA kit Rotavirus VP7 48 tests

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