High-Throughput Centrifugal Microfluidic Nucleic Acid Diagnostic System Based on an Integrated Spatial Encoding Strategy for Simultaneous Detection of Gut Bacteria and Gut Virome

Infectious intestinal disease (IID) poses a significant threat to human health and development due to its high morbidity and mortality rates. However, current diagnostics are challenged by diverse human enteropathogens and difficulties in simultaneously detecting gut bacteria and the virome. Herein, we propose a high-throughput centrifugal microfluidic nucleic acid diagnostic system that couples an integrated spatial encoding strategy with loop-mediated isothermal amplification (LAMP) for the simultaneous detection of 12 human enteropathogens (termed MiLASD) via a novel "nucleic acid extraction-targeted amplification-spatial coding" strategy. The MiLASD system integrates automated Chelex-100 resin-based nucleic acid purification, programmable LAMP reactions, and real-time fluorescence signal analysis into a single CD-shaped microfluidic chip. Positioned in the chip's inner circle, Chelex-100 resin efficiently removes proteins and salts, demonstrating superior sensitivity and faster kinetics than the thermolysis method. Using a single fluorescent dye, the system converts nucleic acid amplification into fluorescence signals while simultaneously detecting up to 48 targets via microfluidic spatial encoding, overcoming the multiplexing limitations inherent to conventional LAMP assays. Additionally, screened multiplex LAMP primer sets enable comprehensive human enteropathogen profiling. Post-optimization, MiLASD achieves detection limits of 10² CFU/mL for bacterial targets and 10 copies/μL for viral targets and completes multiplex identification within 45 min. Qualitative analysis of 125 clinical stool samples showed 100% positive predictive agreement and 100% negative predictive agreement. Requiring neither complex instruments nor specialized operators, MiLASD promises significant potential for enhanced diagnosis and epidemiologic surveillance of human enteropathogens.