Rapid and sensitive detection of the bovine viral diarrhea virus genome in semen.
A reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed for the detection of bovine viral diarrhea virus (BVDV) in cell culture supernatant and in bovine semen. Several sets of primers, PCR conditions and extraction methods were examined to optimize the procedure. A set of primers designed to amplify a highly conserved portion of the p80 gene from BVDV (corresponding to NADL strain sequence from bp 6668 to 7107), was demonstrated to be the most effective. These oligonucleotide primers consistently amplify a 440-bp fragment from several non-cytopathic and cytopathic biotypes of BVDV. The viral origin of the PCR products was assessed by sequencing. The introduction of a Sephacryl S-400 chromatography step to remove seminal inhibitors prior to RNA extraction permitted RT-PCR detection of BVDV in raw and extended semen samples. A maximum sensitivity of 0.4 TCID50 was achieved with this method using RNA extracted from tissue supernatants. This RT-PCR assay may be a useful tool for the detection of BVDV in semen of persistently infected bulls.
Departamento de Patología Animal I, Universidad Complutense de Madrid, Facultad de Veterinaria, Spain.
N
N
Da Silva
R
R
Zardoya
G
G
Santurde
A
A
Solana
J M
JM
Castro
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000302:isolation & purification*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D012661:Semen / Q000821:virology*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral