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

<b>Introduction:</b> 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 <i>Aspergillus</i> spores in clinical and environmental samples. <b>Methods:</b> 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. <b>Results and discussion:</b> The chip achieved a lysis efficiency of 85.5% for <i>Aspergillus</i> 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 <i>Aspergillus</i> <i>fumigatus, Aspergillus flavus</i>, and <i>Aspergillus niger</i>, 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 <i>in vitro</i> diagnostics.