Development of Pentaplex Reverse Transcription Droplet Digital PCR Assay for Simultaneous Detection and Absolute Quantification of HIV‐1, HIV‐2, HCV, and HBV With Internal Control

This study presents the development of a pentaplex reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay for the simultaneous detection and absolute quantification of human immunodeficiency virus (HIV)-1, HIV-2, hepatitis C virus (HCV), hepatitis B virus (HBV), and an internal control. These viruses are important blood-borne pathogens for diagnostics, blood screening, and the validation of reference materials. Commercial kits used for blood screening allow multiplex detection but rely on relative, cycle threshold-based quantification approaches; consequently, the results may vary depending on reference materials and analytical conditions, which limit interlaboratory comparability. In addition, conventional antigen-antibody assays and single-target quantitative polymerase chain reaction (qPCR) methods have limited multiplexing capability and require standard curves, restricting their broader application. The assay demonstrated 95% limit of detection (LoD) values ranging from 2.42 to 6.65 copies/reaction across targets with high specificity. Validation following the principles outlined in International Organization for Standardization (ISO) 20395:2019 demonstrated the linearity, sensitivity, and precision of the assay, with low coefficients of variation indicating high reproducibility. When evaluated using a nanowell plate-based system, the assay showed comparable performance without statistically significant differences, thereby demonstrating platform scalability. The applicability of the assay was further confirmed using externally sourced pathogen resources, including human plasma-derived HIV-1-positive materials under conditions resembling clinical specimens, demonstrating reliable detection beyond international reference materials. This study introduces a robust multiplex platform for diagnostics, blood screening, and quality control of reference materials. The assay integrates qualitative and quantitative analysis within a single workflow, enabling efficient multi-target detection.