Development of an improved real time PCR for the detection of bovine leukaemia provirus nucleic acid and its use in the clarification of inconclusive serological test results

•The real time PCR was developed for detection BLV proviral DNA.•The test showed higher sensitivity than conventional PCR.•The test clarified the BLV status of cattle with inconclusive ELISA results.

With the aim to erradicate Enzootic Bovine Leukosis from Poland, a more sensitive real-time polymerase chain reaction was required and developed to detect proviral Bovine leukaemia virus (BLV) DNA, the causative agent of Enzootic Bovine Leukosis (EBL). This new method proved more sensitive for our needs, than the current protocols available in the public domain. DNA was extracted from peripheral blood leukocytes of 51 cattle, which had given rise to doubtful serological test results by ELISA, and from mesenteric lymph nodes of six cattle that were slaughtered as EBL suspect cases. Additionally, fourteen DNA samples were obtained from animals with a strong BLV antibody response by ELISA. All real-time data were compared to results obtained from three different nested PCR methods. All 14 strongly positive ELISA samples were positive in all PCR tests. The real-time assay in comparison to the conventional PCR methods detected 7.8% (4/51) more specimens positive for BLV nucleic acid and showed a detection limit down to one copy. These observations represent the first report in the value of using a real-time method to help elucidate the disease status of animals when inconclusive ELISA results are obtained in the diagnostic laboratory. Thus, this method should be recommended for use in countries which have implemented an EBL-eradication programme, where a low level of BLV infection is evident.