A novel multiplex RPA/CRISPR-Cas12a integrated biosensor for on-site detection of high-risk HPV genotypes

Accurate genotyping of high-risk human papillomavirus (HR-HPV) at the point of care is critical for global cervical cancer elimination, but its application remains limited by the need for complex equipment and specialized procedures, particularly in resource-limited regions. Here, we develop H-MRC12a—an integrated platform based on degenerate primers and type-specific crRNAs that combines multiplex recombinase polymerase amplification (RPA) with CRISPR-Cas12a trans-cleavage activity—for ultrasensitive detection of eight key HR-HPV genotypes (16, 18, 31, 33, 52, 53, 58, 66). By introducing a degenerate primer strategy coupled with type-specific crRNAs, the system overcomes interference from primer dimer formation and achieves single-copy sensitivity within 50 minutes under isothermal conditions (37°C). Clinical validation demonstrated 100% concordance with qPCR and identified three additional low viral-load positives (Ct >35) that were missed by conventional methods. Crucially, the platform enables visual readout under UV light and eliminates the need for specialized instruments. This ‘broad-spectrum capture and precision typing’ paradigm establishes a versatile framework for multiplexed pathogen detection, advancing accessible molecular diagnostics for global health equity. Herein, we developed the H-MRC12a biosensor, which integrates Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a technologies. The process initiates with specific recognition of targets by primers, continuously generating amplicons to activate the CRISPR/Cas12a system, leading to significant signal amplification. Using degenerate primers targeting conserved regions of HPV L1, it overcomes primer competition in multiplex amplification through optimized kinetics, achieving single-copy detection sensitivity. • The H-MRC12a POCT platform is the first device that can detect 8 types of HR-HPV at the same time, and it can identify as few as 1 copy. This new platform makes home self-testing for HR-HPV possible. • The H-MRC12a POCT platform solves two major problems: primer dimer formation in multiplex RPA and poor sensitivity for low - concentration targets. • Nucleic acid extraction, amplification and result reading take only 50 min, 60% faster than traditional PCR, ideal for POCT.