Akademik Veri Yönetim Sistemi
Cyprus Journal of Medical Sciences, cilt.10, sa.1, ss.69-73, 2025 (ESCI)
BACKGROUND/AIMS: Hepatitis D virus (HDV) is a significant global health problem identified in the 1970s in Hepatitis B virus (HBV) positive patients. HDV is classified under the Kolmioviridae family and has a unique single-stranded negative-sense circular RNA genome. Its replication relies on HBV surface antigens making HBV co-infection essential. HDV infection can occur either simultaneously with HBV or as a superinfection. This virus frequently leads to progressive liver disease, with approximately 70% of cases developing cirrhosis, highlighting the critical need for early diagnosis. This study aims to develop a high sensitivity and specificity quantitative reverse transcription polymerase chain reaction (RT-qPCR) (diagnostic kit for detecting HDV RNA in plasma samples. The newly developed kit is expected to provide reliable diagnosis and facilitate the early detection of HDV, thereby improving clinical outcomes and epidemiological surveillance. MATERIALS AND METHODS: Following the RNA isolation step using the RN easy RNA Purification commercial kit (QIAGEN, Cat. No: 74104), the samples underwent a heat-shock protocol, 95 °C for 10 minutes, followed by rapid freezing at -20 °C to disrupt the secondary structure of the HDV RNA and enhance primer binding efficiency. The single-step RT-qPCR assay was carried out using a specific primer-probe set targeting conserved regions of the HDV genome, along with a human ribosomal protein (RP) gene as an internal control, to validate RNA extraction and the absence of sample degradation. RT-qPCR was performed using the QIAGEN Rotor-Gene Q-5plex device. RESULTS: Developed RT-qPCR diagnostic kit successfully detected HDV RNA in all patient samples with high specificity. The fluorescence signals obtained from both the FAM (HDV target) and HEX (RP gene) channels confirmed the accurate amplification of the target regions. The kit was further validated using blind samples obtained from the Molecular Diagnosis and Quality Control Laboratory to ensure its clinical applicability and robustness. CONCLUSION: The development of a novel HDV-specific RT-qPCR diagnostic kit provides a valuable tool for the early, accurate detection and quantification of HDV RNA in clinical samples. The kit’s ability to offer rapid and reliable results, coupled with its high sensitivity and specificity, makes it an excellent candidate for widespread clinical use and epidemiological monitoring. Further validation studies are recommended to expand its application across diverse clinical settings and to evaluate its performance in different HDV genotypes. Furthermore, the in-house kit that was produced is thought to be a more affordable alternative to the current commercial kits, which might make it more accessible, particularly in low-income nations.