A selective enrichment and specific probe terminal mediated strategy for highly sensitive detection of microRNAs.

MicroRNAs are promising liquid biopsy biomarkers, but their clinical translation is hindered by detection challenges, including low abundance, high sequence similarity, and background interference. Here we present SE-SPTM-PCR, a detection platform integrating selective miRNA enrichment using locked nucleic acid probes with specific probe terminal mediated RT-qPCR. We show that SE-SPTM-PCR eliminates nonspecific amplification and achieves 100-fold higher sensitivity than conventional stem-loop RT-qPCR. In clinical studies, SE-SPTM-PCR significantly improves hsa-miR-92a-3p performance for colorectal cancer detection, increasing its AUC from 0.72 to 0.85 in 48 patients versus 48 controls. Additionally, SE-SPTM-PCR also restores utility to two abandoned biomarkers: For HCMV reactivation monitoring in 32 DNA positive and 32 DNA negative hematopoietic stem cell transplant recipients, hcmv-miR-UL22A-5p achieves an AUC of 0.95. For nasopharyngeal carcinoma, ebv-miR-BART3-3p reaches a perfect AUC of 1.0 in 40 patients and 40 controls. This platform provides a robust tool for miRNA-based liquid biopsy, offering enhanced diagnostic accuracy to support early disease detection and personalized treatment strategies.