Ultra-sensitive One-step Platform for Logic Screening and Detecting of HPV Genotypes via a Programmable Amplification-transcription Cascade

Abstract

Ultra-sensitive, versatile, and contamination-resistant nucleic acid detection remains a major challenge in molecular diagnostics, particularly for applications requiring simultaneous pathogen screening and precise genotyping. Herein, we report a programmable, label-free isothermal sensing strategy termed probe-initiated amplification and transcription for high-sensitivity sensing platform (PATSP), which decouples target recognition from signal generation via a universal signal-transduction architecture. In this system, target-specific hybridization events are converted into a universal intermediate trigger that activates a fixed amplification-transcription cascade, generating malachite green (MG) RNA aptamers as the fluorescent output. By integrating loop-mediated isothermal amplification (LAMP) with in situ transcription in a single closed-tube format, PATSP achieves high sensitivity while effectively suppressing aerosol contamination and false-positive signals. Notably, only minimal probe sequence replacement is required for new targets, enabling one-tube multiplex screening using a single primer pair without primer cross-reactivity. Under optimized conditions, PATSP enables attomolar-level detection of multiple human papillomavirus (HPV) genotypes (11, 16, 18, and 52) and human immunodeficiency virus (HIV) over a wide dynamic range. Clinical validation using cervical secretion samples showed 100% concordance with quantitative polymerase chain reaction (qPCR) for both HPV screening and genotyping. Collectively, PATSP provides a robust and broadly applicable platform for nucleic acid diagnostics and point-of-care testing.

Keywords

Isothermal amplification / Nucleic acid detection / Human papillomavirus / Genotyping / Fluorescent aptamer

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RIGHTS & PERMISSIONS

Jilin University, The Editorial Department of Chemical Research in Chinese Universities and Springer-Verlag GmbH