Standardization of a polymerase chain reaction (Semi Nested–PCR) to detect bovine herpesvirus type 1 in aborted fetus and semen from naturally infected cattle

Abstract

The glycoprotein D gene of bovine herpesvirus type 1 (BHV-1) was detected in clinical samples from naturally infected cattle by semi-nested polymerase chain reaction (SN-PCR). Different protocols were tested to increase the sensitivity and specificity of the technique. An association of DNA extraction methods using phenol/chloroform/isoamyl alcohol followed by silica/guanidine isothiocyanate yield greater concentration and quality of amplified DNA. After optimization of primers and reaction conditions, the genome of BHV-1 (Los Angeles strain) was detected by SN-PCR in tissue culture supernatant and artificially infected semen at the 1 and 0.1 TCID50 limit, respectively. When used on clinical specimens from naturally infected cattle, the SN-PCR yield positive results in semen of seropositive bull and in organ fragments of aborted cattle fetus. The SN-PCR was a viable alternative, which was faster, sensitive, specific and less laborious to be used in the routine diagnosis of BHV-1 infection and semen health monitoring.