J Clin Microbiol. 2009 Apr 1.

New molecular detection tools adapted to emerging rhinoviruses and enteroviruses.

Laboratory of Virology, Division of Infectious Diseases, University of Geneva Hospitals and Faculty of Medicine, University of Geneva, Geneva, Switzerland; Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin-Madison, Wisconsin, USA; Division of Pediatric Pulmonary Medicine, University Hospital of Bern, Bern, Switzerland; Institute of Microbiology, University Hospital of Lausanne, Lausanne, Switzerland; Institute for Infectious Diseases, University Hospital of Bern, Bern, Switzerland.

Human rhinoviruses (HRV), and to a lesser extent human enteroviruses (HEV), are important respiratory pathogens. Similar to 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 peptide nucleic acid-based hybridization oligoprobes (Panenterhino/Ge/08 PNA probe) designed to detect all HRV and HEV species members according to publicly available sequences. The RT-PCR assays have been validated using not only plasmid and viral stocks, but also quantified RNA transcripts and around 1000 clinical specimens. These new generic detection PCR assays overcame the variability of circulating strains, and lowered the risk of missing emerging and divergent human rhino- and enteroviruses. An additional real-time PCR assay (Entero/Ge/08) was also designed specifically to provide a sensitive and targeted detection of HEV in cerebrospinal fluid. In addition to the generic probe, we developed specific probes for the HRV-A and HRV-B detection in cells. This investigation provides a comprehensive toolbox for an accurate molecular identification of the different human entero- and rhinoviruses circulating in humans.

http://jcm.asm.org/cgi/content/abstract/JCM.02339-08v1