An Asymmetric Stem-Loop Translator Enhances Specificity of One-Pot Isothermal CRISPR/Cas12a Assay

Sensitive and specific detection of molecular biomarkers is fundamental to clinical diagnostics and biomedical research, yet existing CRISPR-based assays often suffer from nonspecific activation and cross-reactivity. Here, we introduce an asymmetric stem-loop translator that markedly enhances the specificity of a one-pot isothermal strand displacement amplification (SDA)-CRISPR/Cas12a assay. The asymmetric probe enables precise molecular recognition and controlled signal transduction, converting diverse targets into amplifiable DNA intermediates. Within the integrated one-tube system, SDA-generated DNA products directly trigger Cas12a trans-cleavage, yielding rapid fluorescence responses without thermal cycling or manual intervention. Systematic optimization of reaction parameters effectively mitigated enzyme inhibition and aerosol contamination. The resulting assay achieves highly specific and sensitive detection of miRNA with a detection limit of 500 fM, accurately distinguishing single- and double-base mutations. Owing to its modular design, the asymmetric stem-loop translator is readily adaptable to other analytes. By coupling to an aptamer-based recognition element, the system enables sensitive aflatoxin B1 detection. This work establishes a generalizable framework for enhancing CRISPR/Cas12a specificity through asymmetric molecular translation, offering a versatile platform for rapid nucleic acid and small-molecule diagnostics in clinical and point-of-care settings.