Detection of human respiratory syncytial virus in respiratory samples by LightCycler reverse transcriptase PCR.
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.
Whiley
David M
DM
Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital and Health Service District, Herston, Queensland, Australia.
Syrmis
Melanie W
MW
Mackay
Ian M
IM
Sloots
Theo P
TP
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
D005456:Fluorescent Dyes; D012367:RNA, Viral