A self-contained ternary probe assay integrating self-priming cascade amplification with CRISPR/Cas12a for ultrasensitive detection of infant UTI-related microRNA.

Accurate quantification of infant urinary tract infection (UTI)-associated microRNAs remains challenging due to their short length, low abundance, and high sequence homology. Herein, we present a sensitive fluorescent biosensor that integrates a self-priming extension cascade with CRISPR/Cas12a-mediated signal amplification, with the core innovation being a rationally designed ternary sensing probe that consolidates all recognition and amplification elements into a single, self-contained architecture on magnetic beads. This integrated design eliminates the need for multiple exogenously added primers, offering three inherent advantages: fixed stoichiometric ratios ensure reproducible reaction initiation, the unified format simplifies the operational workflow, and stringent coupling of amplification to target recognition suppresses non-specific background. Upon target recognition, the probe undergoes programmed conformational rearrangements that trigger sequential self-priming extension events, simultaneously generating fragments that drive autocatalytic feedback and T7 promoter-containing products for downstream crRNA transcription. The transcribed crRNA activates Cas12a collateral cleavage, producing exponentially amplified fluorescence. Under optimized conditions, the assay achieves a detection limit of 0.48 fM, excellent single-base discrimination, and reliable performance in human serum. Quantification results correlate strongly with qRT-PCR. This exogenously primer-free, self-contained cascade uniquely unifies signal amplification, target regeneration, and crRNA synthesis in a single probe format, while the seamless coupling with CRISPR/Cas12a trans-cleavage ensures both ultrahigh sensitivity and exceptional specificity. The proposed strategy provides a promising platform for early diagnosis and screening of infant UTI.