Rapid and ultrasensitive detection of telomerase activity in clinical samples using a PCR-free enzymatic assay

Telomerase, a critical biomarker for cancer diagnosis, necessitates the development of convenient, efficient, and highly sensitive detection methods. Distinct from conventional nucleic acid amplification techniques such as PCR, LAMP, or RCA, this method achieves signal amplification through Endo IV-mediated enzymatic turnover. A rationally designed probe containing an abasic site specifically hybridizes with telomerase-synthesized (TTAGGG)n repeats, enabling precise Endo IV recognition and cleavage that separates a fluorophore-quencher pair for fluorescence signal generation. Cyclic signal amplification is achieved through probe displacement-driven enzymatic turnover. Following systematic optimization (abasic site at nucleotide 11, 500 nM probe, 0.1 U/µL Endo IV), the method exhibits a broad linear range (5-2000 cells) and high sensitivity with a detection limit of 5 HeLa cells. It also demonstrates excellent precision (intra-day RSD: 1.31-1.75%; inter-day RSD: 4.60%), high accuracy (spiked recovery: 97.3-102.1%), and robust anti-interference capability in complex matrices. The assay distinguishes telomerase‑positive cancer cells (NB4, K562, A549, MCF-7, HeLa) from telomerase‑negative normal cells (MRC-5), and correlates well with a commercial ELISA kit. In clinical tissue samples, it differentiated gastric carcinoma from adjacent normal tissues. Collectively, this study provides an analytical platform for telomerase activity detection with potential applications in cancer research and diagnostics.