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

Abstract

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.

FIG. 1

(A) Locations and orientations (arrows) of primers used to amplify the 5′ NCRs of 39 rhinovirus isolates and prototypes (see Materials and Methods). The gray bar represents the 5′ NCR from which the nucleotide sequence was determined. The black regions, labeled A to F, indicate the locations of the relatively long, well-conserved subregions. For reference, the complete rhinovirus genome is given at the top. (B) Nucleotide sequences (cDNA) of the six relatively long, well-conserved subregions of the rhinovirus 5′ NCR. At the top, the consensus sequence of each region is given together with its relative position based on the rhinovirus serotype 2 sequence. Identical residues are indicated with dots; a dash indicates that no nucleotide residue is present at that particular position. At the bottom, the sequences of the primers and probes used in the study are given. Arrows indicate primer orientations. The coxprim1 to coxprim4 primers were previously published by Kämmerer et al. (22). The codes of the rhinovirus field isolates and prototypes from which the sequences were derived are given on the left. Rhinovirus serotype x is abbreviated as rhino x. The seven-digit numbers represent field isolates; the first two digits indicate the year of isolation. The sequences of rhinoviruses marked with bullets were downloaded from the GenBank sequence database.

FIG. 2

Dendrogram based on the sequence alignment of a 530-nucleotide region (bordered by regions A and F) of the 5′ NCRs of 44 rhinoviruses. The codes of the rhinovirus field isolates (seven-digit numbers) and prototypes (“rhino x”) from which the sequences were derived are given on the right. ■, sequence obtained from the GenBank database.