One-Tube, Multiplexed, and Dual-Mode Detection of Antibiotic-Resistant Bacteria via Customizable Allosteric DNAzyme

Accurate and timely detection of antibiotic-resistant bacteria is critical for controlling their spread and achieving precision medicine, necessitating the development of sensitive, contamination-free, and rapid approaches. Nucleic acid-cleaving DNAzymes have been extensively exploited in rapid and precise metal ion sensing; however, their featured response to single-stranded DNA/RNA has limited their application in accurate diagnosis of antibiotic-resistant bacteria, as double-stranded DNA (dsDNA) targets are typically used. To address this limitation, we innovatively developed an elongated DNAzyme-integrated asymmetric recombinase polymerase amplification, named EDA, for the one-tube and multiplexed detection of methicillin-resistant <i>Staphylococcus aureus</i>. Benefiting from the synergistic recognition and amplification capabilities of the cascaded recombinase polymerase amplification and DNAzyme catalytic reaction, single-bacterium-level sensitivity was achieved for both <i>nuc</i> and <i>mecA</i> genes in the fluorescent mode and detection limits as low as 10¹ CFU/mL were attained in the colorimetric detection mode. Furthermore, EDA exhibited excellent specificity, accuracy, and detection efficiency, featuring a sample-to-result time of 1 h. The constructed approach broadens the application scope of the DNAzyme and provides a contamination-free, multiplexed, and fully customizable detection platform for antibiotic-resistant bacteria.