Target Recognition Triggered Split DNAzyme based Colorimetric Assay for Direct and Sensitive Methicillin-Resistance Analysis of Staphylococcus aureus

J Microbiol Biotechnol. 2024 Jun 28;34(6):1322-1327. doi: 10.4014/jmb.2404.04012. Epub 2024 Apr 19.

Abstract

The accurate and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant Staphylococcus aureus (S. aureus) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of S. aureus in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the S. aureus concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.

Keywords: G-quadruplex; MRSA; aptamer; split DNAzyme.

MeSH terms

  • Aptamers, Nucleotide*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Biosensing Techniques / methods
  • Colorimetry* / methods
  • DNA, Catalytic* / metabolism
  • Humans
  • Methicillin Resistance
  • Methicillin-Resistant Staphylococcus aureus* / isolation & purification
  • Penicillin-Binding Proteins / genetics
  • Penicillin-Binding Proteins / metabolism
  • Sensitivity and Specificity
  • Staphylococcal Infections* / diagnosis
  • Staphylococcal Infections* / microbiology
  • Staphylococcus aureus / genetics
  • Staphylococcus aureus / isolation & purification

Substances

  • DNA, Catalytic
  • Aptamers, Nucleotide
  • Bacterial Proteins
  • Penicillin-Binding Proteins