RAPID AND SENSITIVE DETECTION OF THE BOVINE VIRAL DIARRHEA VIRUS GENOME IN SEMEN

A reverse transcription-polymerase chain reaction (RT-PCR) procedure w as developed for the detection of bovine viral diarrhea virus (BVDV) i n cell culture supernatant and in bovine semen. Several sets of primer s, PCR conditions and extraction methods were examined to optimize the procedure. A set of primers designed to amplify a highly conserved po rtion of the p80 gene from BVDV (corresponding to NADL strain sequence from bp 6668 to 7107), was demonstrated to be the most effective. The se oligonucleotide primers consistently amplify a 440-bp fragment from several non-cytopathic and cytopathic biotypes of BVDV. The viral ori gin of the PCR products was assessed by sequencing. The introduction o f a Sephacryl S-400 chromatography step to remove seminal inhibitors p rior to RNA extraction permitted RT-PCR detection of BVDV in raw and e xtended semen samples. A maximum sensitivity of 0.4 TCID50 was achieve d with this 50 method using RNA extracted from tissue supernatants. Th is RT-PCR assay may be a useful tool for the detection of BVDV in seme n of persistently infected bulls.