An integrated centrifugal microfluidic CRISPR-based diagnostics platform for multiplexed point-of-care testing of respiratory pathogens.

Rapid, accurate, and multiplexed point-of-care testing (POCT) of respiratory pathogens is critical for clinical triage and infection control. However, existing platforms frequently necessitate trade-offs between sensitivity, throughput, and operational complexity. To address this, we developed the iCARD (integrated Centrifugal Assay on a Rotating Disc) platform, a streamlined microfluidic molecular diagnostic system based on single-step CRISPR kinetics and centrifugal microfluidics. Comprising a polymethyl methacrylate disc pre-loaded with lyophilized CRISPR/Cas13a reagents and a self-developed portable fluorescence analyzer, the platform enables automated multiplexed detection following sample loading. Furthermore, a novel resin-based pretreatment method was engineered to efficiently purify and concentrate nucleic acids from throat swabs. The iCARD system facilitates the parallel screening of six respiratory pathogens across four independent samples within 40 min. Analytical validation confirmed single-copy sensitivity (limit of detection: 0.25-1.0 copies/μL) without cross-reactivity. Clinical validation using 94 retrospective patient throat swabs demonstrated exceptional diagnostic accuracy, achieving a sensitivity of 95.5% (64/67; 95% CI: 87.64%-98.47%) and a specificity of 100.0% (27/27; 95% CI: 87.51%-100.00%). These findings demonstrate that the iCARD platform serves as a robust, high-throughput, and accurate diagnostic tool for decentralized molecular screening and epidemiological surveillance.