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

Area of Science:

• Biotechnology
• Molecular Biology
• Antimicrobial Resistance

Background:

• Accurate detection of antibiotic-resistant bacteria is crucial for effective treatment and infection control.
• Current methods often face limitations in sensitivity, speed, and contamination prevention.
• DNAzymes, while useful for single-stranded targets, are less suited for double-stranded DNA (dsDNA) common in bacterial diagnostics.

Purpose of the Study:

• To develop a sensitive, contamination-free, and rapid detection method for antibiotic-resistant bacteria.
• To overcome the limitations of DNAzymes in detecting dsDNA targets.
• To create a one-tube, multiplexed platform for diagnosing methicillin-resistant Staphylococcus aureus (MRSA).

Main Methods:

• Development of an elongated DNAzyme-integrated asymmetric recombinase polymerase amplification (EDA) technique.
• Integration of recombinase polymerase amplification (RPA) with DNAzyme catalytic activity for synergistic detection.
• Utilized both fluorescent and colorimetric detection modes for gene targets (nuc and mecA).

Main Results:

• Achieved single-bacterium-level sensitivity for nuc and mecA genes.
• Obtained low detection limits of 10^1 CFU/mL in colorimetric mode.
• Demonstrated high specificity, accuracy, and efficiency with a 1-hour sample-to-result time.

Conclusions:

• The EDA approach successfully broadens DNAzyme applications for dsDNA targets.
• Provides a contamination-free, multiplexed, and customizable platform for detecting antibiotic-resistant bacteria.
• Enables rapid and precise diagnostics essential for precision medicine and infection control.