Development of a highly sensitive, high-throughput and automated CRISPR-based device for the contamination-free pathogen detection.

Abstract

Rapid, portable, and contamination-resistant nucleic acid detection methods are necessary due to the threat posed by emerging viruses to public health and agricultural output. We establish CARE (CRISPR-associated airtight real-time electronic diagnostic device), a novel platform that combines CRISPR-Cas12a with a hermetically sealed microfluidic chip to overcome the limitations of present technologies, which struggle to balance sensitivity, multiplexing, and field applicability. By combining isothermal amplification and CRISPR detection within a hermetically sealed microfluidic chip, CARE eliminates the risk of nucleic acid aerosol contamination while enabling simultaneous high-throughput analysis of seven pathogens. The device is complemented by a user-friendly nucleic acid quantification App, enabling rapid and precise analysis. The RPA-CRISPR/Cas12a system demonstrates exceptional sensitivity, detecting as few as 1 copy μL^-1 (single-plex) and 10-10² copies μL^-1 (multiplexed), with real-sample performance matching gold-standard methods. CARE represents a significant advancement in CRISPR-based diagnostics, offering a robust, portable solution for on-site pathogen detection in food and agricultural applications.