Development of rapid and sensitive RPA-CRISPR-Cas12a assay for HPV16 detection to aid in point-of-care cervical cancer screening

Cervical cancer ranks as the fourth most prevalent cancer among women globally, primarily caused by Human papillomavirus (HPV). HPV 16 stands out as the most commonly identified genotype in cervical cancer cases globally. Due to the limited sensitivity of traditional cancer screening tests, molecular HPV diagnostics have been employed as part of early screening for HPV infection. In this study, we developed an HPV 16 detection method that uses recombinase polymerase amplification (RPA) combined with the CRISPR-Cas12a fluorescence system. The results from the RPA-CRISPR-Cas12a fluorescence assay are easily visualized either under ultraviolet light or through real-time fluorescence-based methods. This platform enables highly specific detection of HPV 16, with a remarkable sensitivity down to a single copy using fluorescence. In a study of 80 clinical isolates, the assay demonstrated 100 % sensitivity, highlighting its reliability. Unlike traditional methods, which are time-consuming, the RPA-CRISPR-Cas12a assay delivers results in just 40 min at a steady temperature of 37 °C. The improvised one-pot RPA-CRISPR-Cas12a fluorescence assay delivers visual changes, which is visible to the unaided eye in as little as 20 min, making this technique particularly suitable for point-of-care testing (POCT) in clinical settings. In conclusion, the RPA-CRISPR-Cas12a fluorescence assay is a promising, rapid, and reliable method for HPV 16 detection with strong potential for future POCT applications.