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Journal of Clinical Microbiology, September 2009, p. 2704-2712, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.00378-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization{triangledown}

Leen-Jan van Doorn,1* Bernhard Kleter,1 Evert Hoefnagel,1 Isabelle Stainier,2 Annick Poliszczak,2 Brigitte Colau,2 and Wim Quint1

DDL Diagnostic Laboratory, Fonteijnenburghlaan 7, 2275 CX Voorburg, The Netherlands,1 GlaxoSmithKline Biologicals, Rue de l'Institut 89, Rixensart, Belgium2

Received 19 February 2009/ Returned for modification 6 April 2009/ Accepted 17 June 2009

Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (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 by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with 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 analyses 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 the superiority of the novel method, mainly in cases of mixed rotavirus infections. This novel method permits highly accurate detection and identification of human rotavirus infections in stool samples. This validated assay could be useful for large-scale epidemiological and clinical trials.

* Corresponding author. Mailing address: DDL Diagnostic Laboratory, Fonteijnenburghlaan 7, 2275 CX Voorburg, The Netherlands. Phone: 0031-70-3401670. Fax: 0031-70-3401671. E-mail: L.J.van.Doorn{at}ddl.nl

{triangledown} Published ahead of print on 24 June 2009.

Journal of Clinical Microbiology, September 2009, p. 2704-2712, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.00378-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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]  


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