JCM Accepts, published online ahead of print on 24 June 2009

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J. Clin. Microbiol. doi:10.1128/JCM.00378-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Detection and genotyping of human rotavirus VP4 and VP7 genes by reverse transcriptase PCR and reverse hybridization

Leen-Jan van Doorn, Bernhard Kleter, Evert Hoefnagel, Isabelle Stainier, Annick Poliszczak, Brigitte Colau, and Wim Quint

DDL Diagnostic Laboratory, Fonteijnenburghlaan 7, 2275 CX Voorburg, the Netherlands; GlaxoSmithKline Biologicals, Rue de l'Institut 89, Rixensart, Belgium

Background and aim: 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 on a strip to detect amplimers and identify specific G and P genotypes present in human stools. An additional aim was to permit specific identification of the rotavirus G1[P8] strain, used in the Rotarix^TM vaccine.

Materials and Methods: 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 (RHA) 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 contains type-specific probes for P4, P6, P8, P9 and P10. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8 and G9. In addition, probes were present to discriminate between G1 wild-type and G1 vaccine strain sequences.

Results: Both RT-PCR methods were confirmed to permit detection of a broad spectrum of genotypes, as tested by analysis of multiple reference strains. RT-PCR for VP7 was more sensitive than for VP4, but all rotavirus antigen-positive samples, as determined by ELISA, were also positive for both VP4 and VP7. 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, precision, accuracy and robustness. In a panel of 149 ELISA-positive stool samples, comparison with conventional type-specific RT-PCR methods revealed superiority of the novel method mainly in case of mixed Rotavirus infections.

Conclusion: This novel method permits highly accurate detection and identification of human rotavirus (HRV) infections in stool samples. This validated assay could be useful for large scale epidemiological and clinical trials.

This article has been cited by other articles:

• Pietsch, C., Petersen, L., Patzer, L., Liebert, U. G. (2009). Molecular Characteristics of German G8P[4] Rotavirus Strain GER1H-09 Suggest that a Genotyping and Subclassification Update Is Required for G8. J. Clin. Microbiol. 47: 3569-3576 [Abstract] [Full Text]