Abstract
Respiratory syncytial virus (RSV) is known to cause acute lower respiratory tract infections (ARI) in young children and is involved in exacerbation of chronic obstructive pulmonary disease (COPD) in adults. The role of RSV in stable COPD and the viral load in different respiratory diseases has not been investigated to date. The present authors established and evaluated a quantitative TaqMan real-time polymerase chain reaction assay specific for RSV subgroup A. Absolute quantification for the determination of viral load input was achieved using a control plasmid. The assay allowed for a quantification over a >6-log range and a detection limit of <10 RSV copies per reaction mixture. The assay was 30 times more sensitive than conventional nested polymerase chain reaction assays. Interassay SD was 1.3 and coefficient of variation 4.7% on average. Clinical specimens from infants with ARI (n=62) and elderly adults with COPD (n=125) were compared for viral loads. A total of 47% RSV-positive samples were found in the ARI study group and 28% in the COPD study group. The viral load of both study groups was found to differ significantly. In the ARI study group the viral load was increased almost 2000-fold, suggesting acute infection in this group and former or latent infection in the COPD group. Respiratory syncytial virus-A specific TaqMan real-time polymerase chain reaction assay is a sensitive and rapid method for the determination of viral load in clinical samples. It enables differential statements concerning the involvement of respiratory syncytial virus in acute lower respiratory tract infections and chronic obstructive pulmonary disease to be achieved.
Highlights
Evaluation of a quantitative real-time polymerase chain reaction (PCR) for the detection of respiratory syncytial virus in pulmonary diseases
Respiratory syncytial virus-A specific TaqMan1 real-time polymerase chain reaction assay is a sensitive and rapid method for the determination of viral load in clinical samples. It enables differential statements concerning the involvement of respiratory syncytial virus in acute lower respiratory tract infections and chronic obstructive pulmonary disease to be achieved
The predictive length of the Quantitative real-time PCR (qPCR) amplification product was confirmed by a single clear band of 76 bp length on an ethidium bromide-stained 4% NuSieve1 agarose gel (FCM BioProducts, Rockland, ME, USA) under ultraviolet illumination
Summary
Evaluation of a quantitative real-time PCR for the detection of respiratory syncytial virus in pulmonary diseases. ABSTRACT: Respiratory syncytial virus (RSV) is known to cause acute lower respiratory tract infections (ARI) in young children and is involved in exacerbation of chronic obstructive pulmonary disease (COPD) in adults. The present authors established and evaluated a quantitative TaqMan real-time polymerase chain reaction assay specific for RSV subgroup A. Respiratory syncytial virus-A specific TaqMan real-time polymerase chain reaction assay is a sensitive and rapid method for the determination of viral load in clinical samples. It enables differential statements concerning the involvement of respiratory syncytial virus in acute lower respiratory tract infections and chronic obstructive pulmonary disease to be achieved.
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