Detection and quantitation of bovine respiratory syncytial virus using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays

• Published in: Journal of virological methods 2004-10, Vol.121 (1), p.1-6
• Main Authors: Achenbach, Jenna E., Topliff, Christina L., Vassilev, Ventzislav B., Donis, Ruben O., Eskridge, Kent M., Kelling, Clayton L.
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Bovine respiratory syncytial virus; Cattle; Cell Line; Fluorescence; Fluorescent Dyes - metabolism; Fundamental and applied biological sciences. Psychology; iCycler; Microbiology; Quantitative competitive RT-PCR; Real-time quantitative RT-PCR; Reference Standards; Respiratory Syncytial Virus, Bovine - genetics; Respiratory Syncytial Virus, Bovine - isolation & purification; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - standards; RNA, Messenger - analysis; RNA, Viral - analysis; Sensitivity and Specificity; Techniques used in virology; Time Factors; Virology; Virology - methods
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2004.05.004

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/μl 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.