Development of a Bovine leukemia virus polymerase gene-based real-time polymerase chain reaction and comparison with an envelope gene-based assay

Bovine leukemia virus (BLV) causes a persistent infection with provirus formation in B-lymphocytes. A real-time polymerase chain reaction (PCR) based on the conserved BLV polymerase (BLV pol) gene sequences was developed. Dually labeled probes were used to permit detection by the 5' exonuclease assay. The assay was validated with 350 samples of bovine peripheral blood mononuclear cells including 144 samples from BLV-seropositive animals worldwide (South America, Europe, Middle East, Australia) representing 5 of the recently described 7 BLV envelope-based genotypes. The BLV pol real-time PCR proved to be highly specific and sensitive with the detection of up to 1 copy of an internal control plasmid. The 95% confidence intervals for assay sensitivity and specificity were =98.27% and =98.33%, respectively. Restriction fragment length polymorphism and phylogenetic BLV pol-based sequence analysis of the investigated samples were performed and compared with the previous described BLV env-based genotypes. Grouping of the sequences based on the pol gene yielded similar results as the env gene-based assay.