A duplex real-time RT-PCR for the detection of bluetongue virus in bovine semen

The control measures prescribed by the World Organization for Animal Health (OIE) for international trade in extended semen implicate repeated free testing of the donor's blood for bluetongue virus (BTV). The aim of this study was to validate a real-time RT-PCR for the direct testing of semen for artificial insemination (AI). The amplification of the BTV target was combined with an internal control target in duplex format. Optimal RNA recovery and efficient removal of PCR inhibitors was established using Trizol-based RNA extraction. The total assay was highly repeatable, the preliminary analysis of the specificity was 100% (95% CI: 92-100%) and the limit of detection was -0.36 log(10)TCID(50) ml(-1) (95% CI: -0.53 to -0.18 log(10)TCID(50) ml(-1)) in BTV-8 spiked extended semen. The protocol was evaluated using 89 extended semen samples from 19 bulls showing typical clinical signs of a natural BTV-8 infection. Forty-eight samples were positive, 30 were doubtful and 11 were negative. Infectious BTV-8 was isolated. Based on varying real-time RT-PCR results of additional straws from cut-off samples it is highly recommended to analyse at least five straws per semen batch before declaring semen free of BTV. In conclusion, the partially validated assay presented has the potential to be used for the control of semen for international trade through direct testing of the semen.