title: Detection of human respiratory syncytial virus in respiratory samples by LightCycler reverse transcriptase PCR.
abstract: Laboratory diagnosis of human respiratory syncytial virus (hRSV) infections has traditionally been performed by virus isolation in cell culture and the direct fluorescent-antibody assay (DFA). Reverse transcriptase PCR (RT-PCR) is now recognized as a sensitive and specific alternative for detection of hRSV in respiratory samples. Using the LightCycler instrument, we developed a rapid RT-PCR assay for the detection of hRSV (the LC-RT-PCR) with a pair of hybridization probes that target the hRSV L gene. In the present study, 190 nasopharyngeal aspirate samples from patients with clinically recognized respiratory tract infections were examined for hRSV. The results were then compared to the results obtained with a testing algorithm that combined DFA and a culture-augmented DFA (CA-DFA) assay developed in our laboratory. hRSV was detected in 77 (41%) specimens by LC-RT-PCR and in 75 (39%) specimens by the combination of DFA and CA-DFA. All specimens that were positive by the DFA and CA-DFA testing algorithm were positive by the LC-RT-PCR. The presence of hRSV RNA in the two additional LC-RT-PCR-positive specimens was confirmed by a conventional RT-PCR method that targets the hRSV N gene. The sensitivity of LC-RT-PCR was 50 PFU/ml; and this, together with its high specificity and rapid turnaround time, makes the LC-RT-PCR suitable for the detection of hRSV in clinical specimens.
authors.affiliation: Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital and Health Service District, Herston, Queensland, Australia.
authors.forename: David M
authors.initials: DM
authors.lastname: Whiley
authors.forename: Melanie W
authors.initials: MW
authors.lastname: Syrmis
authors.forename: Ian M
authors.initials: IM
authors.lastname: Mackay
authors.forename: Theo P
authors.initials: TP
authors.lastname: Sloots
mesh_terms: D002648:Child; D002675:Child, Preschool; D031541:Fluorescence Resonance Energy Transfer; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D006801:Humans; D009305:Nasopharynx / Q000821:virology*; D012367:RNA, Viral / Q000032:analysis; 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
chemical_list: D005456:Fluorescent Dyes; D012367:RNA, Viral