Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples

Abstract

Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme Scal, which specifically cleaved products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (890%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.

Acute phase protein changes in calves during an outbreak of respiratory disease caused by bovine respiratory syncytial virus

“…Age of calves at week 0 (days) growth as described by Autio et al [18]. The nested RT-PCR method described by Vilcek et al [19] was used for detection of BRSV and the RT-PCR method [20] for detection of bovine corona virus (BCV).…”

Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds

“…RSV was detected by nested RT-PCR targeting the F fusion protein gene (Vilcek et al, 1994). BCV was detected using primers described by Tsunemitsu et al (1999), and real-time PCR was used to amplify synthesized cDNA (95 8C 1 min, 35 cycles of 96 8C for 10 s, 94 8C for 10 s, 58 8C for 20 s, 72 8C for 20 s, data collected at 81 8C, 72 8C for 10 min).…”

Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples

“…9402022) (Viuff et al, 1996) with a concentration of 10 4.5 TCID 50 /ml. Total RNA was extracted from each dilution and was analysed by fRT-PCR (two replicates of each dilution, from 10 to 10 9 ) and in parallel by nested PCR (Vilcek et al, 1994). The experiment was repeated with both assays.…”

“…The introduction of the sensitive conventional PCR assays for virus diagnosis has lead to a significant improvement in the detection of BRSV (Larsen et al, 1999;Oberst et al, 1993;Valarcher et al, 1999;Vilcek et al, 1994;West et al, 1998). These methods, however, require complex laboratory manipulations and are therefore time-consuming and expensive (Belák and Thorén, 2001).…”