Concanavalin-A-assisted extraction-free one-pot RPA-CRISPR/Cas12a assay for rapid detection of HPV16.

Human papillomavirus (HPV) infection is a major threat to women's health worldwide. High-risk subtypes, particularly HPV16, require rigorous screening and long-term surveillance to control cervical cancer. However, traditional HPV testing is hampered by the need for nucleic acid extraction, reliance on specialized technicians, and fluorescence detection equipment, limiting its suitability for rapid on-site testing. In this study, we developed a Concanavalin A-assisted extraction-free one-pot recombinase polymerase amplification (RPA) CRISPR/Cas12a assay (ConRCA) for HPV16. Concanavalin A-coated magnetic beads were used for target enrichment and nucleic acid-extraction-free processing. Suboptimal protospacer-adjacent motifs were used to achieve a one-pot RPA-CRISPR/Cas12a assay. The ConRCA assay can be completed in approximately 25 min under isothermal conditions and can detect at least 1.2 copies/μL of HPV16 genomic DNA using a fluorescence reader or test strip, demonstrating comparable sensitivity to qPCR. The feasibility of this detection method was evaluated with 31 unextracted clinical samples. Compared with qPCR, the overall sensitivity was 95% (19/20), and the specificity was 100% (11/11). Our results indicate that the ConRCA assay has great potential utility as a point-of-care testing for the rapid identification of HPV.