PNA-assisted magnetic bead-confined CbAgo-CRISPR/Cas12a cascade for amplification-free detection of Mycobacterium avium subsp. paratuberculosis F57 DNA

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne’s disease in ruminants, has attracted increasing attention because of its potential zoonotic relevance. Sensitive molecular detection of MAP typically relies on nucleic acid amplification to overcome low pathogen abundance; however, amplification may generate nonspecific products and false-positive results. Here, targeting MAP-specific F57 double-stranded DNA, we developed a peptide nucleic acid (PNA)-assisted, magnetic bead-confined Clostridium butyricum Argonaute (CbAgo)-CRISPR/Cas12a cascade electrochemiluminescent (ECL) biosensing platform for amplification-free MAP detection. The platform comprises two functional magnetic-bead microreactors. In the PNA-CbAgo cleavage microreactor (PCCM), PNA and CbAgo/gDNA complexes are co-immobilized on magnetic beads. PNA-mediated local duplex opening facilitates CbAgo cleavage and generation of target-derived DNA fragments (tDNA). In the CRISPR/Cas12a-Ru ECL signaling microreactor (CREM), CRISPR/Cas12a-crRNA complexes and Ru-labeled ssDNA reporters are co-confined on magnetic beads. The generated tDNA activates localized CRISPR/Cas12a trans-cleavage, leading to reporter release and a target-dependent decrease in ECL intensity. Mechanistic studies showed that PCCM enhances tDNA generation through PNA-assisted duplex opening and bead interfacial confinement, whereas CREM enables efficient ECL transduction through localized CRISPR/Cas12a-crRNA-reporter coupling. Under optimized conditions, the platform achieved a detection limit of 34.1 CFU mL−1, exhibited good specificity, agreed well with qPCR in spiked bovine fecal samples, and demonstrated robust overall performance in complex matrices. This work provides an amplification-free ECL strategy for sensitive and specific MAP detection and highlights the potential of bead-confined programmable nuclease cascades for pathogen nucleic acid analysis.