Detection of Bovine Viral Diarrhea Virus in Bovine Semen Using Nested-PCR

Abstract

A rapid and sensitive reverse transcription polymerase chain reaction (RT-PCR) and nested-PCR were used to detect bovine viral diarrhea virus 1 (BVDV-1) in bull semen. Selected primers could amplify a part of the 5´UTR of the BVDV genome. A 294 bp DNA fragment was amplified and specificity of the results was confirmed by direct sequencing of the PCR product. Prior to RNA extraction, the seminal inhibitors were eliminated using a simple dilution method. Therefore, a sensitivity of 3×102 50% tissue culture infective dose (TCID50) was achieved when experimentally infected semen was used for RNA extraction. In nested-PCR a 160 bp fragment was amplified and sensitivity of the test was increased to 3 TCID50. This technique can be used as a rapid and sensitive method of BVDV-1 detection in bovine semen.