A direct multiplex isothermal amplification-reverse dot blot hybridization system for β-thalassemia diagnosis

Molecular diagnostic methods largely rely on expensive equipment and complex operations, which makes it difficult to achieve rapid and low-cost detection. In recent years, the increasing demand for point-of-care testing has driven the rapid development of isothermal amplification techniques, due to their simplicity and low equipment requirements. However, complex nucleic acid extraction steps are still required before most isothermal amplification. In this study, we propose a nucleic acid extraction-free gene detection method: direct multiplex Recombinase Aided Amplification combined with reverse dot blot hybridization (dmRAA-RDB). This method can detect multiple targets simultaneously and offers advantages such as high sensitivity, high specificity, low cost, no need for expensive instruments, and visual detection. By pre-treating whole blood samples with sodium hydroxide solution, the samples can be directly used for isothermal amplification and combined with commercial reverse dot blot (RDB) technology to rapidly detect 17 types of β-thalassemia mutations. The experimental results demonstrated that the pre-treated whole blood samples allowed for the simultaneous, stable, and efficient enrichment of all three target fragments via direct multiplex isothermal amplification. This single-tube triple amplification strategy demonstrates significant innovation in the field of isothermal amplification. Moreover, the workflow is shortened by nearly half, and a side-by-side comparison of 60 clinical samples showed 100 % agreement with the commercial PCR-RDB kit. The dmRAA-RDB method offers a highly promising, innovative solution for large-scale, rapid, and low-cost β -thalassemia screening in primary-care and resource-limited settings, and opens a new avenue for detecting other mutant genes.