Multiplex on-chip detection of Aspergillus by integrated ultrasonication-based bead beating lysis and magnetic beads direct amplification

Introduction: Invasive aspergillosis (IA) is a life-threatening disease in immunocompromised individuals, creating an urgent need for rapid, sensitive, and user-friendly methods for early detection of Aspergillus spores in clinical and environmental samples. Methods: In this study, we developed a rapid ultrasonication-based beads beating (USBB) method for cell lysis and fungal DNA release, along with an integrated magnetic beads-based direct amplification (MBDA) method utilizing microfluidic chip technology. Our flexible thin-film microfluidic chip was developed to enable immediate contacting with an ultrasonic oscillator for nucleic acid extraction. Results and discussion: The chip achieved a lysis efficiency of 85.5% for Aspergillus at 10 5 spores per sample. Magnetic beads releasing Fe ions at 75-860 ng/mL allowed high-efficiency direct PCR amplification. Complete transfer of the nucleic acid extract into PCR amplification led to a 100-fold improvement in detection sensitivity. The integrated USBB-MBDA system completed the entire workflow from sample-in to result-out within 35 min (5 min fungal DNA release and 30 min TaqMan assay). Using this approach, we achieved simultaneous triplex detection of Aspergillus fumigatus, Aspergillus flavus , and Aspergillus niger , with a sensitivity as low as 10 spores per test. Validation on 37 clinical samples showed complete concordance with MALDI-TOF mass spectrometry (MS). This study establishes an integrated nucleic acid extraction and magnetic bead–enabled direct amplification strategy, providing a versatile approach for the development of automated analytical platforms with broad applicability to in vitro diagnostics.