A Visual Isothermal Platform for the Rapid Detection of Clostridioides difficile in Fecal Using Locked Nucleic Acid-Modified Split DNAzyme Probes Facilitated by Recombinase Polymerase Amplification

Early, rapid, and accurate diagnosis is of great significance for the timely treatment of C. difficile. This study constructed a visual isothermal analysis platform for C. difficile in feces based on recombinase polymerase amplification (RPA) and locked nucleic acid (LNA)-modified four sets of split DNAzyme probes, which can complete nucleic acid amplification and detection within 40 minutes. RPA technology avoids the need for thermal cycling instruments, and greatly reduces amplification time. The combination of LNA-modified probes and target nucleic acid is extremely stable, and neither temperature nor enzyme influence can dissociate it. The split G-quadruplex/hemin DNAzyme maintains its catalytic capability to oxidize the colorless substrate 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS²⁻) into its green radical form ABTS∙⁻. Of particular significance, LNA-modified split DNAzyme probes with the help of several enzymes in the RPA system can accurate and stable capture of target double-stranded DNA, subsequently facilitating the DNAzyme formation. This innovative approach significantly streamlines the experimental procedure by minimizing operational complexity and reducing processing time. The assay outcomes are readily discernible through direct visual observation and can be conveniently documented using a smartphone. Therefore, this method of visual detection for C. difficile established in this study has the advantages of being time efficient, and having a high sensitivity and specificity.