Development and application of a new method for amplification and detection of human rhinovirus RNA

A method based on nucleic acid sequence based amplification (NASBA) was developed for detection of rhinovirus RNA. Appropriate collection and storage conditions for maintenance of rhinovirus RNA integrity in clinical samples was determined. Two silica-based extraction methods were evaluated for preparation of RNA from virus isolates and clinical samples. Primers and probes were selected from the non-translated region at the 5' end and from VP4 of sequenced rhinoviruses. Amplified products were detected by 'in-solution' hybridization, with analysis by polyacrylamide gel electrophoresis (enzyme linked gel assay or ELGA), and by a microtitre-based plate hybridization assay. Using propagated picornavirus isolates in vitro the rhinovirus NASBA, with detection of amplified sequences by ELGA or plate hybridization, was confirmed as sensitive and specific for detection of rhinovirus RNA. The method was applied successfully to analysis of rhinovirus sequences in clinical samples from individuals with respiratory-tract symptoms. Rhinovirus NASBA will be useful for studies of the molecular epidemiology of respiratory infections and monitoring of response to anti-rhinovirus therapy.