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).

Real-time PCR to improve the diagnosis of respiratory syncytial virus infection

“…It was found that two-step real-time PCR was approximately 25 % more sensitive than nested RT-PCR and 60 % more sensitive than antigen ELISA. This is in agreement with a report of Gueudin et al (2003), which was based on real-time RT-PCR using a LightCycler instrument with primers targeting the RSV N gene. It was shown that real-time RT-PCR is able to detect also low level of virus in clinical samples.…”

Development of a New Method for Diagnosis of Rubella Virus Infection by Reverse Transcription-Loop-Mediated Isothermal Amplification

“…In previous reports, RT-PCR for rubella targeting the E1 region detected up to 8 infectious units of WHO international standard/ml in amniotic fluid (8,15) and RSV (9,12), but real-time PCR for rubella virus has not been reported. This method is based on PCR temperature shifts and shows higher sensitivity than RT-PCR with a reduction of the risk of cross-contamination.…”

Rapid Diagnostic Method for Detection of Mumps Virus Genome by Loop-Mediated Isothermal Amplification

“…It was applied mainly for the retrospective laboratory studies, not as a clinical diagnostic tool. Recently, real-time PCR was introduced for the detection of several pathogens and is now being used for RNA viruses in single-tube real-time RT-PCR for the detection of RNA viruses such as respiratory syncytial virus (3,28). A special apparatus is also required for real-time PCR, but this is beneficial for obtaining virus copies from the samples within a few hours.…”