A Cas12a-based fluorescent microfluidic system for rapid on-site human papillomavirus diagnostics

Area of Science:

• Molecular Diagnostics
• Biotechnology
• Oncology

Background:

• Persistent human papillomavirus (HPV) infection is the primary cause of cervical cancer.
• Early diagnosis of HPV genotypes is essential for effective clinical management.
• Current on-site diagnostic methods for HPV genotyping lack speed and ease of use.

Purpose of the Study:

• To develop a rapid, sensitive, and on-site diagnostic system for detecting six common HPV subtypes.
• To integrate Cas12a-based detection with microfluidics and recombinase polymerase amplification (RPA) for HPV genotyping.
• To validate the performance of the developed system using clinical samples.

Main Methods:

• Screening of crRNAs and RPA primers targeting the HPV L1 gene for sensitive and specific detection.
• Development of a one-pot RPA reaction for simultaneous amplification of six HPV subtypes without cross-reactivity.
• Integration of RPA-Cas12a detection into a microfluidic device for rapid on-site analysis.

Main Results:

• The developed system successfully detected six HPV subtypes (HPV6, 11, 16, 18, 31, 33) with high sensitivity and specificity.
• The one-pot RPA reaction amplified target HPV subtypes effectively without cross-reactivity.
• The microfluidic assay provided results within 35 minutes and demonstrated a 99.1% concordance rate with qPCR using 112 clinical swab samples.

Conclusions:

• The Cas12a-based fluorescent microfluidic system offers a rapid, sensitive, and user-friendly on-site assay for HPV genotyping.
• This technology has significant potential to improve cervical cancer screening and prevention strategies.
• The system's speed and on-site capability address the limitations of current diagnostic approaches.