Identifying Pathogen and Allele Type Simultaneously (IPATS) in a single well using droplet digital PCR
A combined host biomarker and pathogen diagnosis provides insight into disease progression risk and contributes to appropriate clinical decision-making regarding prevention and treatment. In preventive veterinary medicine, such combined diagnosis could improve risk-based livestock herd management. We developed a single-well based test for combined diagnosis of bovine leukemia virus (BLV) and bovine MHC (BoLA)-DRB3 alleles. A fourplex droplet digital PCR method targeting the BLV pol gene, BLV-susceptible DRB3*016:01 allele, resistant DRB3*009:02 allele, and housekeeping RPP30 gene (IPATS-BLV) successfully measured the percentage of BLV-infected cells and determined allele types precisely. Furthermore, it discriminated homozygous from heterozygous carriers. Using this method to determine the impact of carrying these alleles on the BLV proviral load (PVL), we found DRB3*009:02-carrying cattle could suppress the PVL to a low or undetectable level, even with the presence of a susceptible allele. Although the population of DRB3*016:01-carrying cattle showed significantly higher PVLs when compared with cattle carrying other alleles, their individual PVLs were highly variable. Because of the simplicity and speed of this single-well assay, IPATS could be a suitable platform for the combined diagnosis of host biomarkers and pathogens in a wide range of other systems.