Development of multiplex reverse-transcription digital PCR assay for co-detection of bovine leukemia virus and bovine viral diarrhea virus in cattle

Bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) are important transboundary pathogens that cause substantial economic losses in the cattle industry globally. Their early detection and control are critical for preventing disease transmission and minimizing their impact on livestock health and productivity. Therefore, establishing a sensitive and robust diagnostic method capable of simultaneously detecting BLV and BVDV is vital for implementing timely control measures. Here, we developed a multiplex RT-dPCR assay to detect BLV and BVDV in a single-tube reaction using nucleic acid extracted from whole blood of infected cattle. The multiplex RT-dPCR assay successfully detected both BLV and BVDV with high specificity, exhibiting no cross-reactivity with other bovine viruses including Akabane virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, bovine herpesvirus 1, and bovine immunodeficiency virus. The assay demonstrated the ability to detect BVDV-1 and BVDV-2 at minimum titers of 10² and 10 ³ TCID₅₀/mL, respectively. For BLV, the multiplex RT-dPCR assay exhibited a detection limit as low as 18.7 viral copies. Our findings represent a significant advance in the detection of BLV and BVDV from extracted RNA and cDNA, highlighting the potential of assays for achieving improved diagnostic accuracy and early disease intervention.