Cleavable energy transfer labeled oligonucleotide probe for enhanced isothermal amplification detection and nano digital chip-based readout

Quantitative analysis of human papillomavirus (HPV)-infected cervical cancer is essential for early diagnosis and timely treatment of cervical cancer. Here, we introduce a novel energy transfer-labeled oligonucleotide probe to enhance the loop-mediated isothermal amplification (LAMP) assay for highly sensitive and specific detection of HPV 16. Conducted as a single-step assay within a digital nanofluidic chip featuring numerous reaction reservoirs, our method facilitates target amplification under isothermal conditions. Targeting an HPV 16 gene, our chip demonstrates the capability to detect HPV DNA at concentrations as low as 1 fM, spanning a dynamic range of five orders of magnitude. Importantly, our nano digital chip enables for highly quantitative detection at low concentrations, with the correlation between target concentration and the number of microwells exhibiting fluorescence signals. Furthermore, we have developed a computer vision method for automated and 100% accurate quantification of target concentrations. This research holds promising applications in clinical diagnosis and is poised for seamless integration into both hospital and point-of-care settings.