Ultra-Sensitive, Label-Free Detection of Nosocomial Infection RNA Biomarkers via CRISPR-Cas13a-Initiated PER-Driven DNA Walker

Nosocomial infection is a major global cause of mortality, posing significant public health challenges. Recent studies have identified RNA biomarkers, including microRNA (miRNA) and circular RNA (circRNA), as promising diagnostic tools for nosocomial infection, providing essential information for timely clinical intervention. In this study, we developed a versatile and label-free circRNA and miRNA detection platform based on a Cas13a/crRNA complex-based target recognition that triggers a primer exchange reaction (PER)-driven DNA walker. In this method, the Cas13a/crRNA complex is used to specifically identify target RNA biomarkers and to induce subsequent signal amplification strategies following cleavage of uracil (UUU, trU)-containing locked "sensing probe" based on trans-cleavage of Cas13a. Taking the merit of the high specific target recognition capability of Cas13a/crRNA, false signals or unintended circRNA degradation can be greatly avoided. The PER performed on the surface of streptavidin-coated magnetic beads (sMBs) motivated the walking of primer sequences, greatly amplifying fluorescence signals. Based on this design, the proposed method exhibits high sensitivity and precision, with detection limits as low as 0.41 fM for miRNA-21 and 3.12 fM for cZNF292. These findings suggest that this strategy could advance the identification of RNA biomarkers and hold considerable potential for future applications in diagnosing nursing infections.