Integrated CRISPR/Cas12a-Based Duplex Detection Platform for Species Fingerprinting

Abstract

Species fingerprinting is crucial to ensure food safety and human health, which requires a rapid, simple, multiplex, and field-deployable detection technique. In response, a lab-on-a-disc microfluidic chip with CRISPR/Cas12a that integrates target preamplification and signal readout enhanced by tetrahedral DNA frameworks (TDFs) has been developed in this study. This platform automates the workflow from recombinase polymerase amplification (RPA) through signal readout. In addition, a portable heating module was developed specifically for colorimetric detection, providing a complete field-deployable solution. The performance of the CRISPR/Cas12a platform was evaluated by detecting cow’s milk adulteration in buffalo milk. The platform demonstrated high sensitivity, achieving detection limits of 1% (v/v) and 5% (v/v) for fluorescence and colorimetric detection within 1 h, respectively. The results agree well with those from real-time quantitative polymerase chain reaction (qPCR) in real-sample analysis. The integrated CRISPR/Cas12a-based duplex detection platform features high sensitivity and specificity, reaction automation, minimal aerosol contamination risk, and decentralized operation, which demonstrates significant potential for field-deployable species fingerprinting and risk prediction.