Establishment of a novel diagnostic test for Bovine leukaemia virus infection using direct filter PCR

Enzootic bovine leucosis (EBL) is a neoplastic disease of cattle caused by Bovine leukaemia virus (BLV). EBL causes great economic losses, so a fast and reliable diagnostic method is critical for understanding the status of BLV. This will allow us to control BLV infections efficiently and mitigate economic losses. In this study, we established a direct diagnostic test for BLV using dried blood-spotted filter papers without sample pre-treatment. The study was based on 159 clinical blood specimens collected in EDTA from one farm in Kyushu, Japan. The blood-spotted filter papers were used as the template for direct filter PCR. When an ELISA was used as the diagnostic gold standard, the sensitivity and specificity of the direct filter PCR were 90.1% and 97.5%, respectively. The kappa value for the direct filter PCR and real-time PCR methods was 0.97. The dried blood samples spotted onto filter papers were stable for at least 10 days at room temperature, even when the samples were from cattle with a low BLV proviral load. Direct filter PCR is a rapid, easy, reliable and cost-effective diagnostic test that directly detects the BLV proviral genome in clinical blood specimens without DNA extraction. Moreover, it simplifies the collection, transportation and storage procedures for clinical blood specimens.