Tehran University of Medical Sciences Acta Medica Iranica 0044-6025 64 9 2026 08 19 594 603 Mandana Zafari Department of Midwifery, Sar.C., Islamic Azad University, Sari, Iran. AND Health Reproductive Research Center, Sari.C., Islamic Azad University, Sari, Iran Taher Sadeghi Department of Histology, Faculty of Science, Urmia University, Urmia, Iran. Sara Banihashemi Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran . Amirreza Hashemi School of Medicine, Ruhr University Bochum, Bochum, Germany . Maryam Bakhtiyari Department of Medical Laboratory Sciences, Faculty of Allied Medicine, Qazvin University of Medical Sciences, Qazvin, Iran. AND Network of Immunity in Infection, Malignancy & Autoimmunity (NIIMA), Universal Scientific Education & Research Network (USERN), Tehran, Iran. Mehrnoush Kowsaryan Thalassemia Research Center, Hemoglobinopathy Institute, Mazandaran University of Medical Sciences, Sari, Iran. Pooria Gill Thalassemia Research Center, Hemoglobinopathy Institute, Mazandaran University of Medical Sciences, Sari, Iran. AND Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Hadis Musavi Thalassemia Research Center, Hemoglobinopathy Institute, Mazandaran University of Medical Sciences, Sari, Iran 2025 11 23 2026 02 18 β-thalassemia is one of the most common autosomal recessive single-gene disorders, particularly in high-prevalence regions. This study aimed to explore the feasibility and diagnostic performance of polymerase chain reaction-denaturing high-performance liquid chromatography (PCR-DHPLC) for non-invasive prenatal detection of the IVS-II-1 (G>A) mutation in fetal DNA. Fifty pregnant women at risk of carrying a fetus with β-thalassemia major were enrolled. Cell-free DNA was extracted from maternal plasma and analyzed using high-resolution melting (HRM) real-time PCR, followed by PCR amplification. Amplicons were assessed by electrophoresis, purified using a PCR purification kit, and subsequently analyzed by DHPLC under predefined buffer and temperature conditions to detect the IVS-II-1 (G>A) mutation. DHPLC analysis identified 13 true positive, 11 true negative, 19 false negative, and 7 false positive cases compared with the invasive reference method. The calculated sensitivity and specificity were 40.6% and 61.1%, respectively, indicating limited diagnostic performance in the current setting. PCR-DHPLC does not provide sufficient sensitivity and specificity for reliable non-invasive prenatal detection of the IVS-II-1 (G>A) mutation in β-thalassemia major. Further methodological optimization and extended mutation panels in larger cohorts are necessary to assess its potential for clinical validation. https://acta.tums.ac.ir/index.php/acta/article/view/11898 https://acta.tums.ac.ir/index.php/acta/article/download/11898/6104