Real Time RT-PCR for the detection and quantitation of bovine respiratory syncytial virus.
A quantitative Real Time RT-PCR assay was developed to detect and quantify bovine respiratory syncytial virus (BRSV) in the respiratory tract of infected animals. A pair of primers and a TaqMan probe targeting conserved regions of the nucleoprotein gene of BRSV were designed. The detection limit of the assay was shown to be 10(3) RNA copies and standard curve demonstrated a linear range from 10(3) to 10(8) copies as well as an excellent reproducibility. The efficiency of the BRSV Real Time RT-PCR was then assessed by detecting BRSV in lungs, tracheas and bronchoalveaolar fluids (BAL) samples of experimentally infected calves. The assay was shown to be 100 times more sensitive than conventional RT-PCR and was more efficient for BRSV diagnosis. Finally, the Real Time RT-PCR was used to quantify BRSV load in BAL fluids of four experimentally infected calves for 14 days. The high sensitivity, rapidity and reproducibility of the BRSV Real Time RT-PCR make this method suitable for diagnostic and for the evaluation of the efficiency of new vaccines.
Boxus
M
M
Virology Department, Veterinary and Agrochemival Research Center, 1180 Uccle, Belgium.
Letellier
C
C
Kerkhofs
P
P
D000818:Animals; D002417:Cattle; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D018112:Respiratory Syncytial Virus, Bovine / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
D012333:RNA, Messenger; D012367:RNA, Viral