High-throughput, sensitive, and field-deployable nucleic acid detection is essential for timely pathogen surveillance. Here, we developed a novel amplification-free and portable platform based on an instrument-free polydisperse digital droplet CRISPR/Cas12a (DD-Cas12a) assay, enabling the simultaneous detection of three synthetic nucleic acid targets in a single reaction. The platform achieved attomolar-level sensitivity (0.15 fM) without pre-amplification, generated detection results within 40 min, and maintained high specificity even in the presence of a 10-fold excess of non-target nucleic acids. The DD-Cas12a was further applied to detect one DNA virus, porcine circovirus type 2 (PCV2), and two RNA viruses, porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV). The detection limits for PCV2, PEDV, and PRRSV were 126.0, 673.5, and 584.5 fg/μL, respectively, corresponding to sensitivity comparable to qPCR for PCV2 and 10-fold higher sensitivity for PEDV and PRRSV. To improve analytical throughput and objectivity, we established an Algorithm-Driven DD-Cas12a (ADC) platform for automated image analysis and result decoding. The custom-designed miniaturized portable instrument was developed for signal acquisition and field deployment. By combining the DD-Cas12a assay with the ADC platform and the portable instrument, we established a portable, digital, amplification-free, and ultrasensitive system for multiplex pathogen detection, providing a powerful tool for diagnostics, biosurveillance, and environmental monitoring.
