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) wa s developed for detection of rhinovirus RNA. Appropriate collection an d storage conditions for maintenance of rhinovirus RNA integrity in cl inical samples was determined. Two silica-based extraction methods wer e evaluated for preparation of RNA from virus isolates and clinical sa mples. Primers and probes were selected from the non-translated region at the 5' end and from VP4 of sequenced rhinoviruses. Amplified produ cts were detected by 'in-solution' hybridization, with analysis by pol yacrylamide gel electrophoresis (enzyme linked gel assay or ELGA), and by a microtitre-based plate hybridization assay. Using propagated pic ornavirus isolates in vitro the rhinovirus NASBA, with detection of am plified sequences by ELGA or plate hybridization, was confirmed as sen sitive and specific for detection of rhinovirus RNA. The method was ap plied successfully to analysis of rhinovirus sequences in clinical sam ples from individuals with respiratory-tract symptoms. Rhinovirus NASB A will be useful for studies of the molecular epidemiology of respirat ory infections and monitoring of response to anti-rhinovirus therapy. (C) 1998 Elsevier Science B.V. All rights reserved.