DETECTION OF BOVINE HERPESVIRUS-1 IN BOVINE SEMEN BY A NESTED PCR ASSAY

A nested PCR assay targeting a portion of the glycoprotein IV gene has been developed for the detection of Bovine Herpesvirus-1 (BHV-1). Rap id and sensitive detection of the PCR products was achieved using a no nisotopic reverse dot-blot format with a visible color readout. Cross- reactivity of this PCR assay was not observed with the closely related BHV-3. The sensitivity of this assay when tested on a supernatant fro m a BHV-1 cell culture was approximately 4.5 TCID50 (50% tissue cultur e infectious dose). A procedure using the chelating resin Chelex 100 w as used to prepare viral DNA from artificially inoculated samples of e xtended and raw semen for use in the PCR assay. In combination with ne sted PCR and reverse dot blot, this method allowed the detection of 5 x 10(3) TCID per 0.5 ml of semen, which is comparable to the detection in the Cornell Semen Test. The whole procedure can be completed in ap proximately 8 h. This assay has therefore the potential of replacing t he currently available yet time consuming and costly detection methods for BHV-I in bovine semen.