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

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Oncology

Background:

• Cervical cancer is a leading cause of death among women globally.
• Persistent high-risk human papillomavirus (hrHPV) infection is the primary cause of cervical cancer.
• Current screening methods are often slow, labor-intensive, and require extensive infrastructure, limiting their use in resource-limited settings.

Purpose of the Study:

• To develop a novel, rapid, and sensitive nucleic acid detection platform for hrHPV.
• To overcome the limitations of existing cervical cancer screening technologies.
• To enable frequent and accessible HPV testing, particularly in underserved regions.

Main Methods:

• Introduction of droplet digital-enhanced Pyrococcus furiosus Argonaute detection (ddPfAgo), a platform combining Argonaute (Ago) nucleic acid detection with digital assay principles.
• Utilizing the heat-activated cleavage activity of PfAgo for selective double-stranded DNA recognition without PAM dependence.
• Employing vortex-assisted droplet generation and an ImageJ-based workflow for multiplexed HPV16 and HPV18 detection within 30 minutes.

Main Results:

• Achieved femtomolar-level detection sensitivity (50 fM limit), a 10^6-fold improvement over traditional solution-based PfAgo methods.
• Demonstrated rapid, multiplexed detection of HPV16 and HPV18 in under 30 minutes.
• Clinical validation showed 100% consistency with quantitative PCR (qPCR), confirming high diagnostic accuracy.

Conclusions:

• ddPfAgo is a novel nucleic acid detection platform with high sensitivity and speed for hrHPV detection.
• The platform eliminates the need for complex microfluidics and advanced computational resources, making it suitable for resource-limited settings.
• ddPfAgo shows significant potential for accurate and accessible clinical diagnosis of cervical cancer.