Development and Validation of a Novel FRET-Based IFPP Assay for Hepatitis B Virus Detection in a Diverse Population

Background Hepatitis B, a major global health concern, necessitates precise diagnostic methods to manage and mitigate its impact effectively. Traditional real-time PCR techniques, while sensitive, face challenges like false positives and susceptibility to viral mutations. This study introduces a novel Forster Resonance Energy Transfer (FRET)-based approach, the Internal Fluorophore Primer-Primer (IFPP) FRET assay, designed to enhance diagnostic accuracy by optimizing the fluorophore and quencher distances in the assay setup. By doing so, it aims to increase sensitivity and specificity, reduce false results, and improve overall hepatitis B detection capabilities. Materials and Methods In the development of the IFPP FRET assay, molecular probes and quenchers are strategically utilized to optimize the detection sensitivity. The assay employs a novel approach where the primers are designed to ensure that the fluorophores and quenchers are placed at a calculated distance, optimizing energy transfer efficiency and reducing the likelihood of quenching anomalies. The diagnostic efficiency of this method is rigorously compared with traditional techniques, emphasizing improvements in sensitivity and specificity due to the refined detection dynamics provided by FRET technology. Results The IFPP Assay demonstrated superior performance compared to the Chip Based Assay with a sensitivity of 96.92% vs. 90.91%, specificity of 95.71% vs. 90.24%, PPV of 95.45% vs. 89.74%, and NPV of 97.10% vs. 91.36%. The assay validation included 110 samples with an additional 160 samples in a prospective study, confirming the assay's robustness and reliability in clinical settings in (Fig 1 & 2 and Table 1). Conclusion The novel IFPP FRET Assay shows significant improvements in sensitivity and specificity over traditional methods. It presents a promising alternative for the early and accurate detection of HBV, which is crucial for effective management and treatment strategies.