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Quantitation of respiratory syncytial virus RNA in nasal aspirates of children by real-time RT-PCR assay.

ABSTRACT: A method was developed for the quantitation of respiratory syncytial virus (RSV) based on real-time RT-PCR using a LightCycler instrument. A control real-time RT-PCR was undertaken on GAPDH mRNA (a human housekeeping gene) was carried out to standardise the non-homogeneous respiratory samples. The real-time RT-PCR method was one log more sensitive for the detection of RSV according to the endpoint dilution technique than the culture method or a conventional qualitative RT-PCR-hybridization-EIA. No cross-reactivity was observed with any of the viruses that could be found in the respiratory tract. RSV and GAPDH were quantified in nasal aspirates from 75 children hospitalised for acute respiratory tract disease: 31 (41.3%) were positive according to the immunofluorescence assay (IFA), 34 (45.3%) were culture-positive and 42 (56%) were positive according to our real-time RT-PCR method. The sensitivity, specificity, positive and negative predictive values of the real-time RT-PCR were 100, 90, 92, 100%, respectively. The samples found to be positive for RSV were classified according to the severity of the disease. The mean number of RSV RNA copies was higher in the severe disease group than in the non-severe group 4.05 x 10(7) vs 9.1 x 10(6) (P=0.055). However, the mean ratio of RSV RNA copies to GAPDH mRNA copies was 42.8 in the severe group, and 22.2 in non-severe group (P=NS).

SUBMITTER: Gueudin M

PROVIDER: S-EPMC7119593 | biostudies-literature | 2003 Apr

REPOSITORIES: biostudies-literature

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Quantitation of respiratory syncytial virus RNA in nasal aspirates of children by real-time RT-PCR assay.

Gueudin M M Vabret A A Petitjean J J Gouarin S S Brouard J J Freymuth F F

Journal of virological methods 20030401 1

A method was developed for the quantitation of respiratory syncytial virus (RSV) based on real-time RT-PCR using a LightCycler instrument. A control real-time RT-PCR was undertaken on GAPDH mRNA (a human housekeeping gene) was carried out to standardise the non-homogeneous respiratory samples. The real-time RT-PCR method was one log more sensitive for the detection of RSV according to the endpoint dilution technique than the culture method or a conventional qualitative RT-PCR-hybridization-EIA. ...[more]

PMID: 12668266

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Project description:BackgroundBoth respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are important viral pathogens causing respiratory tract infection (RTI) in the pediatric population. However, the clinical manifestations of RSV and hMPV infections are similar. Therefore, a reliable and rapid diagnostic tool is needed for diagnostic performance.MethodsIn order to optimize diagnosis efficiency of RTI, the aim of this study is to establish a rapid and advanced method for simultaneous detecting RSV and hMPV in nasopharyngeal aspirates specimens from patients. We designed a one-step triplex real-time RT-PCR (qRT-PCR) protocol using TaqMan probes for detecting RSV and hMPV. The plasmid clones containing RSV nucleoprotein gene and hMPV fusion gene were established as reference standards. We used virus culture supernatants from 86 known pediatric RTI patient to test the specificity and sensitivity of our assay. Then we used total 222 nasopharyngeal aspirates specimens from pediatric patients hospitalized with respiratory symptoms to evaluate our assay.ResultsOur one-step triplex qRT-PCR assay showed 100% sensitivity and specificity in testing RSV and hMPV in 86 known virus culture supernatants, with excellent linearity (R2 > 0.99) and reliable reproducibility (CV lower than 1.04%). This assay has a wide dynamic range 102-109copies/reaction (limit of detection; LOD = 100 copies/reaction). A total of 222 patients hospitalized with respiratory symptoms were enrolled for clinical evaluation. In these samples, our qRT-PCR assay detected 68 RSV positive and 18 hMPV positive cases. However, standard virus culture only detected 8 RSV positive cases and 0 hMPV cases. Based on this improved triplex qRT-PCR assay, we found that RSV infection was associated with severe inflammation by chest X-ray and occurrence of pneumonia which were not observed previously.ConclusionsIn summary, we have developed a highly specific and sensitive one-step triplex qRT-PCR assay to detect hMPV and RSV simultaneously. This assay offers a valuable tool for routine diagnosis.

Dataset Information

Quantitation of respiratory syncytial virus RNA in nasal aspirates of children by real-time RT-PCR assay.

Publications

Quantitation of respiratory syncytial virus RNA in nasal aspirates of children by real-time RT-PCR assay.

Similar Datasets

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