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

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 10⁵ 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.