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Journal of Clinical Microbiology, June 2009, p. 1742-1749, Vol. 47, No. 6
0095-1137/09/$08.00+0     doi:10.1128/JCM.02339-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

New Molecular Detection Tools Adapted to Emerging Rhinoviruses and Enteroviruses{triangledown}

Caroline Tapparel,1* Samuel Cordey,1 Sandra Van Belle,1 Lara Turin,1 Wai-Ming Lee,2 Nicolas Regamey,3 Pascal Meylan,4 Kathrin Mühlemann,5 Francesca Gobbini,1 and Laurent Kaiser1

Laboratory of Virology, Division of Infectious Diseases, University of Geneva Hospitals and Faculty of Medicine, University of Geneva, Geneva, Switzerland,1 Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin—Madison, Madison, Wisconsin,2 Division of Pediatric Pulmonary Medicine, University Hospital of Bern, Bern, Switzerland,3 Institute of Microbiology, University Hospital of Lausanne, Lausanne, Switzerland,4 Institute for Infectious Diseases, University Hospital of Bern, Bern, Switzerland5

Received 5 December 2008/ Returned for modification 5 February 2009/ Accepted 24 March 2009

Human rhinoviruses (HRV), and to a lesser extent human enteroviruses (HEV), are important respiratory pathogens. Like other RNA viruses, these picornaviruses have an intrinsic propensity to variability. This results in a large number of different serotypes as well as the incessant discovery of new genotypes. This large and growing diversity not only complicates the design of real-time PCR assays but also renders immunofluorescence unfeasible for broad HRV and HEV detection or quantification in cells. In this study, we used the 5' untranslated region, the most conserved part of the genome, as a target for the development of both a real-time PCR assay (Panenterhino/Ge/08) and a peptide nucleic acid-based hybridization oligoprobe (Panenterhino/Ge/08 PNA probe) designed to detect all HRV and HEV species members according to publicly available sequences. The reverse transcription-PCR assay has been validated, using not only plasmid and viral stocks but also quantified RNA transcripts and around 1,000 clinical specimens. These new generic detection PCR assays overcame the variability of circulating strains and lowered the risk of missing emerging and divergent HRV and HEV. An additional real-time PCR assay (Entero/Ge/08) was also designed specifically to provide sensitive and targeted detection of HEV in cerebrospinal fluid. In addition to the generic probe, we developed specific probes for the detection of HRV-A and HRV-B in cells. This investigation provides a comprehensive toolbox for accurate molecular identification of the different HEV and HRV circulating in humans.

* Corresponding author. Mailing address: Laboratory of Virology, Division of Infectious Diseases, University of Geneva Hospitals, 24 Rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. Phone: 41 22 3724085. Fax: 41 22 3724097. E-mail: caroline.tapparel{at}hcuge.ch

{triangledown} Published ahead of print on 1 April 2009.

Journal of Clinical Microbiology, June 2009, p. 1742-1749, Vol. 47, No. 6
0095-1137/09/$08.00+0     doi:10.1128/JCM.02339-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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