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

被引:19
作者
Achenbach, JE
Topliff, CL
Vassilev, VB
Donis, RO
Eskridge, KM
Kelling, CL [1 ]
机构
[1] Univ Nebraska, Dept Vet & Biomed Sci, Lincoln, NE 68583 USA
[2] Univ Nebraska, Dept Biometry, Lincoln, NE 68583 USA
关键词
real-time quantitative RT-PCR; quantitative competitive RT-PCR; iCycler; bovine respiratory syncytial virus;
D O I
10.1016/j.jviromet.2004.05.004
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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(TM). 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/mul 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. (C) 2004 Elsevier B.V. All rights reserved.
引用
收藏
页码:1 / 6
页数:6
相关论文
共 19 条
[1]   Development of a real time quantitative PCR assay for detection of porcine endogenous retrovirus [J].
Argaw, T ;
Ritzhaupt, A ;
Wilson, CA .
JOURNAL OF VIROLOGICAL METHODS, 2002, 106 (01) :97-106
[2]   Detection of bovine viral diarrhea virus in formalin fixed paraffin embedded tissue sections by real time RT-PCR (Taqman) [J].
Bhudevi, B ;
Weinstock, D .
JOURNAL OF VIROLOGICAL METHODS, 2003, 109 (01) :25-30
[3]   Fluorogenic RT-PCR assay (TaqMan) for detection and classification of bovine viral diarrhea virus [J].
Bhudevi, B ;
Weinstock, D .
VETERINARY MICROBIOLOGY, 2001, 83 (01) :1-10
[4]  
Brodersen BW, 1998, AM J VET RES, V59, P1423
[5]   Use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virus [J].
Callahan, JD ;
Brown, F ;
Csorio, FA ;
Sur, JH ;
Kramer, E ;
Long, GW ;
Lubroth, J ;
Ellis, SJ ;
Shoulars, KS ;
Gaffney, KL ;
Rock, DL ;
Nelson, WM .
JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION, 2002, 220 (11) :1636-1642
[6]   Quantitative real-time PCR detection of Rift Valley fever virus and its application to evaluation of antiviral compounds [J].
Garcia, S ;
Crance, JM ;
Billecocq, A ;
Peinnequin, A ;
Jouan, A ;
Bouloy, M ;
Garin, D .
JOURNAL OF CLINICAL MICROBIOLOGY, 2001, 39 (12) :4456-4461
[7]   Comparative evaluation of indirect and sandwich ELISA for the detection of antibodies to bovine respiratory syncytial virus (BRSV) in dairy cattle [J].
Hazari, S ;
Panda, HK ;
Kar, BC ;
Das, BR .
COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES, 2002, 25 (01) :59-68
[8]   THE GENOME OF RESPIRATORY SYNCYTIAL VIRUS IS A NEGATIVE-STRANDED RNA THAT CODES FOR AT LEAST 7 MESSENGER-RNA SPECIES [J].
HUANG, YT ;
WERTZ, GW .
JOURNAL OF VIROLOGY, 1982, 43 (01) :150-157
[9]   BOVINE RESPIRATORY SYNCYTIAL VIRUS-SPECIFIC MONOCLONAL-ANTIBODIES [J].
KLUCAS, CA ;
ANDERSON, GA .
VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY, 1988, 18 (04) :307-315
[10]   CHARACTERIZATION OF BOVINE RESPIRATORY SYNCYTIAL VIRUS PROTEINS AND MESSENGER-RNAS AND GENERATION OF CDNA CLONES TO THE VIRAL MESSENGER-RNAS [J].
LERCH, RA ;
STOTT, EJ ;
WERTZ, GW .
JOURNAL OF VIROLOGY, 1989, 63 (02) :833-840