Detection and quantitation of bovine respiratory syncytial virus using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays.
A single tube, fluorogenic probe-based, real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assay was developed for detection and quantitation of bovine respiratory syncytial virus (BRSV) using BioRad's iCycler iQ. Real-time Q-RT-PCR was compared with quantitative competitive RT-PCR (QC-RT-PCR) and viral titers. Viral mRNA levels were measured in BRSV-infected bovine turbinate cell lysate harvested at eight time points (1.5, 6, 12, 24, 36, 48, 60, 72 h) post-infection. A homologous BRSV cRNA standard was used for quantitation of the mRNA by plotting a standard curve of cycle threshold (Ct) values versus standard 10-fold dilutions of cRNA of known concentrations. Detection as low as 171 copies/microl of standard BRSV cRNA was possible. For QC-RT-PCR, a competitor RNA molecule having a deletion was designed and used for quantitation of the BRSV viral mRNA. The results of real-time Q-RT-PCR and QC-RT-PCR assays showed a positive correlation. Real-time Q-RT-PCR was a sensitive, specific, rapid, and efficient method that eliminates the post-PCR processing steps when compared to QC-RT-PCR. Quantitation of BRSV using real-time Q-RT-PCR will have application in studies aimed at understanding the pathogenesis of BRSV.
Achenbach
Jenna E
JE
Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583-0905, USA.
Topliff
Christina L
CL
Vassilev
Ventzislav B
VB
Donis
Ruben O
RO
Eskridge
Kent M
KM
Kelling
Clayton L
CL
D000818:Animals; D002417:Cattle; D002460:Cell Line; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D012015:Reference Standards; D018112:Respiratory Syncytial Virus, Bovine / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013997:Time Factors; D014773:Virology / Q000379:methods*
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D012367:RNA, Viral