Rapid and reliable detection of G6PD mutations using recombinase polymerase amplification coupled with lateral flow strip

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.