One-tube, protospacer adjacent motif-free and AI-enhanced CRISPR/Cas12a platform for ultra-sensitive detection of human papillomavirus 16 double-stranded DNA

Cervical cancer, primarily caused by HPV16 infection, remains a major global health concern. While PCR is the gold standard for HPV DNA detection, its limitations include lengthy analysis and equipment dependency. To address these challenges, we developed an asymmetric semi-nested RPA combined with Lambda-assisted Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a CRISPR/Cas12a (ASN-RPA-LCRISPR) for protospacer adjacent motif (PAM) independent, ultrasensitive Human Papillomavirus 16 (HPV16) dsDNA detection. This method employs a three-primer asymmetric amplification strategy combined with Lambda nuclease and the CRISPR/Cas12a system to achieve signal amplification. This approach significantly increases single-stranded DNA yield while overcoming PAM restriction issues, achieving a detection limit of 11.8 aM within 35 min. A single-tube system designed in this study prevents aerosol contamination, while a DenseNet121-based image classifier enables 97% accurate fluorescence interpretation under ambient light, eliminating reliance on specialized equipment. This platform offers rapid, ultrasensitive, and portable HPV16 detection, advancing cervical cancer screening and PAM-free nucleic acid diagnostics.