DEVELOPMENT OF A RT-PCR TEST COUPLED WITH A MICROPLATE COLORIMETRIC ASSAY FOR THE DETECTION OF A SWINE ARTERIVIRUS (PRRSV) IN BOAR SEMEN

Transmission of porcine reproductive and respiratory syndrome virus (P RRSV) through boar semen has been demonstrated, stressing the need for a reliable semen PRRSV detection test. A diagnostic assay was develop ed based on amplification of the PRRSV RNA by reverse transcription an d polymerase chain reaction (RT-PCR) followed by detection of the ampl ification products by hybridization and colorimetric assay in microwel l plates. A highly reproducible and efficient method of viral RNA isol ation from semen samples was set up. A combined RT-PCR procedure was p erformed, incorporating the use of uracil-N-glycosylase (UNG) in combi nation with dUTP instead of dTTP to prevent false positive results due to carry-over contamination. An RNA internal control was added during the RNA isolation procedure to detect false negative results. The col orimetric detection in microwell plates of amplification products from either PRRSV or IC RNA gave specific and objective results and was au tomated. A cut-off value of 1000 RNA copies or 10 TCID50 of PRRSV per ml of semen samples could be detected with this assay. Semen samples c ollected from experimentally-infected boars were tested with this assa y and showed PRRSV excretion early after infection and for an extended period. (C) 1997 Elsevier Science B.V.