Rapid Bacterial Identification through Multiplexed Nucleic Acid Detection on a Digital Microfluidic Platform for Enhanced Clinical Intervention against Infections

Abstract

Bacterial infections often lead to severe health consequences owing to their ability to infiltrate multiple anatomical sites, including the bloodstream, respiratory tract, and digestive tract, posing substantial diagnostic and therapeutic challenges. Consequently, a rapid and versatile detection method capable of identifying a broad spectrum of bacterial pathogens is urgently required to facilitate precise antibiotic prescriptions. Addressing this need, we introduce MiND-DMF (Multibacterial Infection Nucleic Acid Detection on a Digital Microfluidic Platform), a cost-effective digital microfluidic platform tailored for multiplexed bacterial detection. This system integrates DNA extraction, recombinase polymerase amplification (RPA), and CRISPR-based detection technologies, enabling the efficient identification of six common infectious bacteria. Operating at a constant temperature of 37 °C, MiND-DMF completes the entire diagnostic process in just 55 min and is compatible with human reference genes. In spiked samples, the platform demonstrated a detection limit of 100 CFU/mL, highlighting its exceptional sensitivity and quantification capability. In clinical evaluations, MiND-DMF exhibited outstanding performance, achieving 100% sensitivity and 98%–100% specificity compared to conventional PCR methods across 50 samples derived from diverse tissue sources. This robust platform demonstrates strong anti-interference capabilities, making it suitable for analyzing various tissue fluids including blood, alveolar lavage fluid, urine, nasal secretions, appendiceal pus, and ear pus. The versatility and precision of MiND-DMF support the monitoring of hospital-acquired bacterial infection origins, empowering physicians to prescribe targeted antibiotics and enhancing overall infection prevention and control strategies. By accurately detecting bacteria from multiple sources, MiND-DMF can play a pivotal role in improving patient outcomes and public health.