Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples.
Bovine respiratory syncytial virus (BRSV) causes severe disease in naive cattle of all ages and is a common pathogen in the respiratory disease complex of calves. Simplified methods for rapid BRSV diagnosis would encourage sampling during outbreaks and would consequently lead to an extended understanding of the virus. In this study, a BRSV fluorogenic reverse transcription PCR (fRT-PCR) assay, based on TaqMan principle, was developed and evaluated on a large number of clinical samples, representing various cases of natural and experimental BRSV infections. By using a single-step closed-tube format, the turn-around time was shortened drastically and results were obtained with minimal risk for cross-contamination. According to comparative analyses, the detection limit of the fRT-PCR was on the same level as that of a nested PCR and the sensitivity relatively higher than that of a conventional PCR, antigen ELISA (Ag-ELISA) and virus isolation (VI). Interspersed negative control samples, samples from healthy animals and eight symptomatically or genetically related viruses were all negative, confirming a high specificity of the assay. Taken together, the data indicated that the fRT-PCR assay can be applied to routine virus detection in clinical specimens and provides a rapid and valuable tool in BRSV research.
Hakhverdyan
Mikhayil
M
Department of Virology, The National Veterinary Institute, Ulls väg 2B, 751 89 Uppsala, Sweden. mikhayil.hakhverdyan@sva.se
Hägglund
Sara
S
Larsen
Lars-Erik
LE
Belák
Sándor
S
D000818:Animals; D002417:Cattle; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D018112:Respiratory Syncytial Virus, Bovine / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D005456:Fluorescent Dyes; D019914:Taq Polymerase