Abstract
Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a common neoplastic disease in cattle. Since animals with high BLV proviral load (PVL) pose a greater transmission risk, identifying cattle with low PVL is crucial for herd management. Certain bovine leukocyte antigen (BoLA)-DRB3 alleles- DRB3*009:02, DRB3*014:01:01, and DRB3*002:01-are strongly associated with low PVL in Holstein cattle and thus can serve as markers for genetic resistance. In this study, we developed a rapid, cost-effective allele-specific PCR (AS-PCR) assay using sequence-specific primers to detect these three alleles. The assay was validated through both synthesized DNA and genomic DNA samples from pre-genotyped cattle, showing 100% sensitivity and 94% specificity compared with PCR-sequence-based typing (SBT). The detection limit was 0.8 ng/reaction. A two-stage pooling approach allowed high-throughput screening of field samples, and analysis of 444 cattle revealed that 27.0% carried at least one target allele. Considering only standard PCR and agarose gel electrophoresis are required, this assay is accessible to basic laboratories without advanced equipment or bioinformatics expertise. Moreover, it enables rapid genotyping of BLV resistance markers, offering a practical tool for genetic selection and disease control in dairy farms, especially those in high-BLV-prevalence regions.