T7-assisted special rolling circular amplification platform for point-of-care cervical cancer screening

Cervical cancer, primarily caused by high-risk human papillomavirus (HPV) infections, remains a leading cause of mortality among women in underdeveloped and developing countries. Conventional screening methods, necessitating professional equipment and trained personnel, are impractical in resource-limited settings, underscoring the need for a point-of-care (POC) detection platform. Isothermal nucleic acid amplification techniques (NAATs) are widely used in POC applications due to their cost-effectiveness and instrument-free nature. In this study, a novel one-pot dual amplification system, T7-MSSRCA was developed, by integrating the minimum secondary structure rolling circular amplification (MSS-RCA) with T7 Exonuclease. This system achieved an amplification time of 15 min and a total diagnostic time of approximately 1 h. Moreover, signal detection using both fluorescence and lateral flow assay (LFA) strips was achieved through reporter probe modifications. By employing a human chorionic gonadotropin (hCG)-modified reporter probe, the T7-MSSRCA system facilitated quantitative POC detection via pregnancy test strips. The system demonstrated impressive sensitivities for synthesized HPV16 targets, achieving 1 fM with fluorescent output and 10 fM with paper strips. When tested with 50 cervical swab samples, T7-MSSRCA exhibited a sensitivity of 0.95 and a specificity of 0.9333 compared to RT-PCR measurements.