Quantitation of respiratory syncytial virus RNA in nasal aspirates of children by real-time RT-PCR assay.
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).
Gueudin
M
M
Laboratory of Human and Molecular Virology, University Hospital, CHR Clémenceau, Av G Clémenceau, 14033 Caen, France.
Vabret
A
A
Petitjean
J
J
Gouarin
S
S
Brouard
J
J
Freymuth
F
F
D000208:Acute Disease; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D006801:Humans; D017817:Nasal Lavage Fluid / Q000821:virology*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D018357:Respiratory Syncytial Virus Infections / Q000821:virology*; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000302:isolation & purification*; D012141:Respiratory Tract Infections / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014776:Virus Cultivation / Q000379:methods
D012333:RNA, Messenger; D012367:RNA, Viral; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)