Improved detection of bovine herpesvirus 1 in artificially infected bovine semen by protein amplification

Use of Epitope Mapping To Identify a PCR Template for Protein Amplification and Detection by Enzyme-Linked Immunosorbent Assay of Bovine Herpesvirus Type 1 Glycoprotein D

“…The reverse primers were designed to contain the Streptag sequence (lowercase) (19) and XhoI restriction site (italic) with primer B located from nucleotide positions 997 to 1013 (5Ј CTC GAG tca acc gaa ctg cgg gtg acg cca agc gct CC GTC GCC TTC GGG TCC 3Ј) and primer D located from nucleotide positions 1315 to 1335 (5Ј CTC GAG tca acc gaa ctg cgg gtg acg cca agc gct CCC GGG CAG CGC GCT GTA GTT 3Ј). For the in vitro transcription and translation reactions, forward primer C was designed to contain T7 RNA polymerase promoter sequence with an ATG codon (5Ј GTA AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AG GG ATG GCC CGG GAT TAC GA 3Ј) and the reverse primer D was designed without the Streptag sequence and XhoI restriction enzyme site (5Ј TCA CCC GGG CAG CGC GCT GTA GTT 3Ј) (29). The BHV-1 DNA preparation and PCR procedure used to obtain specific amplification of the gD gene DNA were described previously (29).…”

“…For the in vitro transcription and translation reactions, forward primer C was designed to contain T7 RNA polymerase promoter sequence with an ATG codon (5Ј GTA AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AG GG ATG GCC CGG GAT TAC GA 3Ј) and the reverse primer D was designed without the Streptag sequence and XhoI restriction enzyme site (5Ј TCA CCC GGG CAG CGC GCT GTA GTT 3Ј) (29). The BHV-1 DNA preparation and PCR procedure used to obtain specific amplification of the gD gene DNA were described previously (29). The PCR products were purified from low-melting-point agarose gels (5) and then cloned into the TA cloning vector pCRII (Invitrogen Life Technologies).…”

“…The detection of BHV-1 in bovine semen is a long-standing problem in veterinary virology which is important in disease control schemes. None of the methods developed so far have been found wholly satisfactory for general use in diagnostic laboratories, especially when applied to semen donor bulls (29). Under these circumstances, our laboratory developed a new method of virus detection consisting of a protein amplification assay following PCR of the BHV-1 gD gene (29).…”

“…None of the methods developed so far have been found wholly satisfactory for general use in diagnostic laboratories, especially when applied to semen donor bulls (29). Under these circumstances, our laboratory developed a new method of virus detection consisting of a protein amplification assay following PCR of the BHV-1 gD gene (29). As the gD polypeptide obtained in the protein amplification assay resembles the gD expressed in Escherichia coli in the absence of glycosylation, a panel of MAbs specific to E. coli-expressed gD was also raised (14).…”

Herpesvírus bovino tipo 1: Tópicos sobre a infecção e métodos de diagnóstico

“…Com freqüência são realizadas amplificações de regiões conservadas dos genes que codificam: i) a glicoproteína B (VILCEK et al, 1994;MASRI et al, 1996;MWEENE et al, 1996;SANTURDE et al, 1996;KATARIA et al, 1997;ROCHA et al, 1998); ii) a glicoproteína C (VAN ENGELENBURG et al, 1993;KATARIA et al, 1997); iii) a glicoproteína D (WEIDMANN et al, 1993;GEE et al, 1996;WAGTER et al, 1996;ZHOU et al, 1999); e iv) a timidina kinase (KIBENGE et al, 1994;YASON et al, 1995;MOORE et al, 2000).…”