Innovative Rapid Detection System for Nondeletion-Type α-Thalassemia via Whole-Blood Amplification and Microfluidic Technology

Thalassemia represents a major global public health challenge. This study developed an integrated detection system combining direct PCR-(Dir-PCR) with reverse dot blot hybridization (RDB), enabling accurate and rapid diagnosis of nondeletion α-thalassemia (α^WSα, α^CSα, α^QSα). The core innovation lies in the systematic optimization of the Dir-PCR-RDB methodology, establishing an efficient amplification and hybridization system capable of directly utilizing whole blood samples, thereby effectively bypassing conventional nucleic acid extraction steps. The optimized method tolerates approximately 24% (12 μL/50 μL) whole blood, achieves a sensitivity of 1.5 copies/μL, and demonstrates no cross-reactivity with other thalassemia variants. Clinical validation showed 100% concordance with sequencing results. Furthermore, by integrating this optimized system with microfluidic technology, we constructed a closed-tube "sample-in, result-out" platform that reduces total detection time to 122 min. This work not only provides an efficient and cost-effective solution for nondeletion α-thalassemia screening but also establishes a technological paradigm for rapid molecular diagnosis of other genetic variants and pathogens.