A high sensitivity-nested PCR assay for BHV-1 detection in semen of naturally infected bulls.
Several different PCR protocols for the detection of Bovine herpesvirus-1 (BHV-1) in bovine semen, are available in the literature. Most of them are rather laborious and the majority were performed on laboratory samples, artificially contaminated semen or semen provided from experimentally inoculated animals. Furthermore, to obtain higher levels of sensitivity, additional dot-blot procedures are frequently necessary. We describe the detection of BHV-1 in bovine semen and the supernatant of cell cultures with titres of 0.001 TCID50/50 microliter by a nested PCR assay, with no further hybridization procedures. The high sensitivity was achieved by filtering the semen samples on chromatography columns before DNA extraction, by using two pairs of primers in a nested PCR and by evaluating the amplification products on silver-stained polyacrylamide gels. Specificity of the amplified fragments was confirmed by RFLP and sequence analysis of the PCR products. This nested PCR procedure was performed in parallel with viral isolation (VI) on 101 semen samples provided from naturally infected bulls housed at an artificial insemination centre. The nested PCR was shown to be more sensitive, faster and easier to perform than the standard VI test. To our knowledge, it is the most sensitive PCR test for BHV-1 detection in bovine semen and could be easily used for routine diagnosis.
Rocha
M A
MA
Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. rochama@dedalus.lcc.ufmg.br
Barbosa
E F
EF
Guimarães
S E
SE
Dias Neto
E
E
Gouveia
A M
AM
D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology; D002478:Cells, Cultured; D017931:DNA Primers; D004279:DNA, Viral / Q000302:isolation & purification; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D007242:Herpesvirus 1, Bovine / Q000235:genetics / Q000302:isolation & purification*; D007316:Insemination, Artificial, Heterologous / Q000592:standards / Q000662:veterinary; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods; D012661:Semen / Q000821:virology*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral