Harnessing CRISPR-Cas12 and Microfluidics Chips for Multiplex Respiratory Pathogens Diagnosis

Respiratory pathogens jeopardize population health, particularly high-risk groups. CRISPR-Cas systems, as novel nucleic acid detection platforms, offer timely identification and have become a major research focus. This study presents a novel diagnostic workflow that combines recombinase polymerase amplification (RPA) for pre-amplification of pathogen nucleic acids with CRISPR-based detection. By combining microfluidic technology and portable imaging devices, this study developed a multiplex assay capable of simultaneously detecting seven clinically relevant pathogens in a single sample, including influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (HRSV) A and B, mycoplasma pneumoniae (MP), adenovirus (HAdv), and parainfluenza virus (HPIVs). Utilizing the POCT-CRISPR platform, simultaneous detection of seven respiratory pathogens can be achieved within approximately 30 min, achieving detection limits of 0.1-1 fM. This method streamlines the detection process, significantly reducing both the complexity of operations and the overall detection time. Clinical cohort validation demonstrated a detection efficiency of 99.63% sensitivity and 100% specificity. These results confirm the effectiveness and reliability of the detection method. Additionally, the 7-virus panel is estimated at approximately $32 per sample, a cost competitive with commercial multiplex qPCR detection kits ($15-$110 per sample) and substantially more economical than integrated cartridge-based syndromic platforms. The platform features simple operation, cost-effectiveness, short turnaround time, and reliable detection performance, making it highly suitable for point-of-care testing (POCT) at the grassroots level.