An integrated microfluidic sample-to-answer system for automated detection of common deletional α-thalassemia mutations.

Thalassemia is a prevalent hereditary hematological disorder, and rapid, automated genetic screening is essential for large-scale prevention and control, particularly in primary healthcare settings. To address the limitations of conventional molecular diagnostic methods, we developed an integrated microfluidic sample-to-answer system for simultaneous detection of four common deletional α-thalassemia mutations (-α3.7, -α4.2, --SEA, and --THAI), together with the HBA2 wild-type locus, enabling accurate discrimination between heterozygous and homozygous deletion genotypes. The system directly uses whole blood without nucleic acid extraction and integrates direct multiplex PCR (dmPCR) with reverse dot blot (RDB) hybridization in a fully closed microfluidic chip, enabling highly automated sample-to-result analysis within 3.5 h while minimizing contamination risk. The RDB-dmPCR assay demonstrated high analytical performance, including a limit of detection of 0.1 μL of whole blood, excellent specificity without cross-reactivity with non-deletional α- or β-thalassemia, strong reproducibility, and robust anti-interference capability. Clinical validation using 97 samples for the RDB-dmPCR assay and an independent cohort of 40 samples for the integrated microfluidic platform achieved 100% sensitivity, 100% specificity, and complete concordance with the reference gap polymerase chain reaction (Gap-PCR) method. By integrating amplification, hybridization, and result interpretation into a single automated workflow, this platform simplifies molecular testing and provides a practical solution for decentralized thalassemia screening in primary healthcare and other resource-limited settings.