Thermally Unlocked One-Pot RPA-CRISPR Cas12b Assay Integrated with the Centrifugal Microfluidic Chip for Multiplex...

Highly contagious porcine viruses, represented by African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), and pseudorabies virus (PRV), inflict severe economic losses on the swine industry and pose significant threats to global food security. Consequently, developing rapid, convenient, and efficient point-of-care testing (POCT) methods is essential for viral disease control. Although recombinase polymerase amplification (RPA) coupled with CRISPR/Cas systems demonstrates significant POCT potential, its practical application is currently restricted by operational complexity and limited throughput. Herein, a thermally unlocked one-pot RPA-CRISPR Cas12b assay integrated with the centrifugal microfluidic chip (TORCH) platform was developed in this paper. In this strategy, a thermal gating switch was utilized to physically isolate CRISPR reagents from the RPA during the initial phase, effectively addressing the inherent incompatibility in one-pot reactions. By employing the centrifugal microfluidic chip with a portable device, a highly integrated workflow enables fully automated processing ranging from sample lysis to multiplexed detection. Validated using pseudovirus-spiked porcine blood samples, TORCH successfully achieved multiplexed detection of ASFV, PRV, and PRRSV with the limits of detection as low as 0.5 copies/μL, while exhibiting exceptional resistance to interference and robust reagent stability. Overall, TORCH stands as a robust and user-friendly diagnostic solution, holding significant potential for early warning intervention and decentralized biosecurity control in resource-scarce environments.