Molecular beacons are a novel class of oligonucleotide probe capable of reporting the accumulation of target amplicon during real-time PCR by the emission of a fluorescent signal. A novel assay for the detection and estimation of respiratory syncytial virus (RSV) nucleic acid in clinical specimens based on real-time PCR utilising such a probe was developed. The probe consisted of two short arm sequences and a central loop sequence complementary to a region of the N gene (the target amplicon). The probe was characterised and a semi-quantitative nested real-time PCR using a LightCycler instrument was optimised. Standard curves were generated using cycle threshold (C-t) values calculated from several assays over a range of logarithmic RSV titres. Linear coefficient correlations were close to one (r(2) = 0.998) and the detection limit of the optimised assay was reproducibly shown to be 1 x 10(-4) pfu/ml. The intra-assay coefficient of variation (CV) of C-t values of the optimal assay was 0.8% and the CV of quantification data was 6.6%. The interassay CV of C-t values was 2.0% and the quantification CV was 6.7%. The validity of the assay for the detection of RSV in clinical specimens was assessed by analysing ten specimens previously assayed in a different laboratory. Real-time PCR analysis was completely consistent with the results of prior analysis. The rapidity, sensitivity and specificity of the assay should greatly facilitate epidemiological studies, particularly in adults as existing methods perform better on clinical specimens from children. (C) 2004 Elsevier B.V. All rights reserved.