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 3x102 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