Cas13a/crRNA trans-cleavage triggered primer exchange reaction based self-priming chain extension for sensitive and label-free infantile pneumonia related microRNA analysis

Abstract

Accurate and sensitive detection of microRNA (miRNA) is crucial for both pathophysiological studies and clinical diagnostics. Conventional amplification methods often face limitations such as dependence on thermal cycling, susceptibility to contamination, and insufficient specificity to discriminate among closely related miRNA family members. To address these challenges, we developed a label-free isothermal detection platform that integrates the precise RNA-targeting ability of the CRISPR/Cas13a system with a self-priming amplification cascade driven by primer exchange reaction (PER). In this assay, target miRNA binding directly activates the trans-cleavage activity of the Cas13a/crRNA complex, which subsequently cleaves a uracil-rich toehold region on a stem-loop DNA primer (H1). Following dephosphorylation, the cleaved primer initiates a PER-mediated self-priming amplification process, generating long tandem double-stranded DNA products that can be sensitively detected using the fluorescent dye SYBR Green I. The proposed method demonstrates several key advantages: (i) high specificity enabled by the programmable Cas13a/crRNA complex, allowing clear distinction between target miRNA and sequences with single-base mismatches or high homology; (ii) exceptional sensitivity, achieving a detection limit of 406 aM and a dynamic range spanning six orders of magnitude, through coupling Cas13a collateral cleavage with exponential isothermal amplification; (iii) excellent reproducibility, reflected by low relative standard deviations and a coefficient of variation of 3.65% in spiked serum samples; and (iv) strong concordance with the reference RT‑qPCR method in mock clinical specimens, highlighting its reliability for potential clinical use. In summary, this CRISPR/Cas13a-coupled self-priming amplification strategy provides a robust, accurate, and highly sensitive means for miRNA quantification, offering a promising alternative for point-of-care molecular diagnostic applications.