Jacob Beatriz Aira C, Songsungthong Warangkhana, Leartsakulpanich Ubolsree, Boonyuen Usa
Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400 Thailand.
Biosensing and Bioprospecting Research Group, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120 Thailand.
Clin Chim Acta. 2025 Jun 15;574:120345. doi: 10.1016/j.cca.2025.120345. Epub 2025 May 3.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy, affecting approximately 500 million people worldwide. It results from inherited mutations in the G6PD gene, causing increased susceptibility to drug-induced hemolytic anemia and severe neonatal jaundice. While phenotypic tests are commonly used, genetic testing is increasingly recognized for its value in the accurate diagnosis of G6PD deficiency, especially in heterozygous females and newborns. This study aimed to develop and evaluate a rapid, field-deployable genetic test for the detection of four common G6PD variants in Thailand: G6PD Gaohe (c.95A > G), G6PD Mahidol (c.487G > A), G6PD Viangchan (c.871G > A), and G6PD Canton (c.1376G > T). The assays utilize recombinase polymerase amplification with allele-specific primers incorporating locked nucleic acids to enhance specificity, followed by lateral flow strip detection for visual readout. The assays deliver results within 45 min at 37 ˚C. Singleplex detection demonstrated 100 % diagnostic sensitivity (Confidence interval (CI): 95.01-100.0 %) and specificity (CI: 95.49-100.0 %). Duplex assays (Gaohe + Canton and Mahidol + Viangchan) also demonstrated 100 % diagnostic sensitivity (CI: 94.87-100.0 %) and specificity (CI: 91.19-100.0 %). Limits of detection (LOD) for singleplex assays were 0.25, 1.00, 0.50, and 0.50 ng/µL, for Gaohe, Mahidol, Viangchan, and Canton, respectively. Duplex assays showed LODs of 0.10 ng/μL for Mahidol + Viangchan and 10.00 ng/μL for Gaohe + Canton. Band intensity differences ranged from 5.25 to 19.61 pixels between mutant, wild-type, and nontarget alleles, enabling clear allele discrimination. This innovative diagnostic tool offers a rapid, reliable, and accessible solution for point-of-care genetic testing, with the potential to improve clinical management and healthcare outcomes in regions with a high burden of G6PD deficiency.
葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症是最常见的酶病，全球约有5亿人受其影响。它由G6PD基因突变遗传导致，使人对药物性溶血性贫血和严重新生儿黄疸的易感性增加。虽然表型检测常用，但基因检测在G6PD缺乏症的准确诊断中的价值日益得到认可，尤其是在杂合子女性和新生儿中。本研究旨在开发并评估一种快速、可现场部署的基因检测方法，用于检测泰国四种常见的G6PD变体：G6PD高河（c.95A＞G）、G6PD马希多尔（c.487G＞A）、G6PD万象（c.871G＞A）和G6PD广州（c.1376G＞T）。这些检测方法利用重组酶聚合酶扩增，使用掺入锁核酸的等位基因特异性引物来提高特异性，随后通过侧向流动条检测进行视觉读数。检测在37˚C下45分钟内出结果。单重检测显示诊断敏感性为100%（置信区间（CI）：95.01 - 100.0%），特异性为100%（CI：95.49 - 100.0%）。双重检测（高河 + 广州和马希多尔 + 万象）也显示诊断敏感性为100%（CI：94.87 - 100.0%），特异性为100%（CI：91.
