S. Vilcek et al., DEVELOPMENT OF NESTED PCR ASSAYS FOR DETECTION OF BOVINE RESPIRATORY SYNCYTIAL VIRUS IN CLINICAL-SAMPLES, Journal of clinical microbiology, 32(9), 1994, pp. 2225-2231
Two nested PCR assays were developed for the detection of bovine respi
ratory syncytial virus (BRSV). Primers were selected from the gene enc
oding the F fusion protein (PCR-F) and the gene encoding the G attachm
ent 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 d
etecting 0.1 tissue culture infective dose of the virus. The PCR-F ass
ay amplified all bovine strains and one human strain (RS32) tested. No
cross-reactions were observed, with nine heterologous respiratory vir
uses. PCR-F products of bovine and human RSV strains were discriminate
d by using endonuclease restriction enzyme ScaI, which specifically cl
eaved products of BRSV. Oligonucleotide probe F was also specific for
products of BRSV. The PCP-G assay detected all bovine strains and none
of the human strains tested. A faint electrophoretic band was also ob
served with products of Sendai virus. However, probe G did not hybridi
ze with this product, only with products of BRSV. Nasal swabs collecte
d from cattle with no symptoms and cattle in the acute stage of respir
atory disease were analyzed for BRSV by the immunofluorescence (IF) me
thod and by the PCR-F and PCR-G assays. The virus was detected by the
PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) wer
e positive by the IF method, and these samples were also positive by b
oth the PCR-F and PCR-G assays. The 31 samples detected as positive by
PCR originated from cattle presenting clinical signs of acute respira
tory disease; the four PCR-negative samples originated from clinically
asymptomatic neighboring cattle. All sampled animals subsequently ser
oconverted and became reactive to BRSV. Thus, the detection of BRSV by
PCR correlated with clinical observations and was considerably more s
ensitive (66 versus 89%) than IF. These results indicate that both nes
ted 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.