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Journal of Clinical Microbiology, March 1999, p. 524-530, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Improved Detection of Rhinoviruses in Clinical Samples by Using a Newly Developed Nested Reverse Transcription-PCR Assay

Arno C. Andeweg,* Theo M. Bestebroer,dagger Martijn Huybreghs, Tjeerd G. Kimman, and Jan C. de Jongdagger

Research Laboratory for Infectious Diseases (LIO), National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Received 12 August 1998/Returned for modification 28 September 1998/Accepted 8 December 1998

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 were 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 extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved 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-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, 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.

* Corresponding author. Present address: Department of Virology, Erasmus University Rotterdam, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. Phone: (31)10 408 80 66. Fax: (31)10 436 51 45. E-mail: andeweg{at}viro.fgg.eur.nl.

dagger Present address: Department of Virology, Erasmus University Rotterdam, Rotterdam, The Netherlands.

Journal of Clinical Microbiology, March 1999, p. 524-530, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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