Improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-PCR assay

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples . The nucleotide sequences of the 5' noncoding regions of 39 rhinoviruses w ere determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA e xtraction method and amplicon identification with probe hybridization to di scriminate between rhinoviruses and the closely related enteroviruses. It p roved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fo ld-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory diseas e patients who had consulted their general practitioners, the new assay dem onstrated a rhinovirus in 24% of the specimens, including all culture-posit ive sample, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.