Transcription cascade reaction-driven isothermal amplification for ultrasensitive detection of high-risk human papillomavirus

Rapid and ultrasensitive nucleic acid detection is crucial for reducing spatiotemporal disparities in clinical outcomes. High-risk human papillomavirus (HPV), which is predominantly linked to cervical cancer, requires early and accurate detection for timely medical intervention. Here, we developed R-transcription cascade reaction (TCR), an isothermal detection platform that integrates recombinase polymerase amplification (RPA) with TCR at 37 °C for the rapid and ultrasensitive detection of HPV 16 and HPV 18, both of which are major oncogenic HPV types. R-TCR amplifies RPA products through TCR, generating light-up RNA aptamers, enabling highly specific and amplified fluorescence detection within 40 min for singleplex and 45 min for multiplex assays. The system reaches an exceptional detection limit of 1 aM (21 copies) in both single-target and multiplex formats, without cross-reactivity. It also enables reliable detection of high-risk HPV DNA integrated into the genomic DNA of cervical cancer cells. In clinical samples, R-TCR achieved 100 % sensitivity and specificity in both singleplex and multiplex assays. These results validate the strong potential of R-TCR as a rapid, ultrasensitive, and economical molecular diagnostic tool for high-risk HPV screening, making it especially suitable for use in low- and middle-income countries.