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

Dec 2, 2025Analytica chimica acta

New combined test for on-site detection of high-risk HPV types

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Abstract

An integrated platform enables 100% concordance with qPCR for detecting eight key high-risk HPV genotypes in under 50 minutes.

The platform combines multiplex recombinase polymerase amplification with CRISPR-Cas12a for sensitive detection.
Single-copy sensitivity is achieved within 50 minutes at 37 °C, overcoming limitations of traditional methods.
Clinical validation identified three additional low viral-load positives that conventional methods missed.
The system allows for visual readout under UV light and does not require specialized equipment.
This approach could facilitate accessible molecular diagnostics in resource-limited settings.

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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 min 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.

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

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