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

Jenna E. Achenbach, Christina L. Topliff, Ventzislav B. Vassilev, Ruben O. Donis, Kent M. Eskridge, Clayton L. Kelling

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1-6
Number of pages6
JournalJournal of Virological Methods
Volume121
Issue number1
DOIs
StatePublished - Oct 1 2004

Keywords

  • Bovine respiratory syncytial virus
  • Quantitative competitive RT-PCR
  • Real-time quantitative RT-PCR
  • iCycler

ASJC Scopus subject areas

  • Virology

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