Multiplexed PfAgo-Based Digital Platform for Clinical Testing of High-Risk HPV Subtypes

Cervical cancer is one of the most common malignant tumors in women. Persistent high-risk human papillomavirus (hrHPV) infection is the cause of nearly all cervical cancers. Routine cervical cancer screening is the most impactful intervention to prevent their progression to invasive disease, but current methods are time-consuming, are labor-intensive, rely on a complex laboratory infrastructure, and are unsuitable for frequent deployment in resource-limited settings. Here, we introduce droplet digital-enhanced Pyrococcus furiosus Argonaute detection (ddPfAgo), a novel nucleic acid detection platform that combines Ago nucleic acid detection with a digital assay. ddPfAgo utilizes the heat-activated cleavage activity of PfAgo to achieve highly selective and efficient double-stranded target DNA recognition without relying on PAM. Through vortex-assisted droplet generation and an ImageJ-based data analysis workflow, ddPfAgo enables the multiplexed detection of HPV16 and HPV18 rapidly within 30 min, without the need for complex microfluidic devices and advanced computational resources. Harnessing the absolute quantification capability of a digital platform, the ddPfAgo achieved detection sensitivity at the femtomolar level (detection limit of 50 fM), representing a 10⁶-fold improvement over traditional solution-based PfAgo methods. Clinical validation confirmed ddPfAgo's high diagnostic accuracy, with test results being 100% consistent with qPCR, demonstrating its great application potential in clinical diagnosis.