Sensitive and Extraction-Free Detection of Methicillin-Resistant Staphylococcus aureus through Ag⁺ Aptamer-Based Color Reaction

Abstract

Refractory infections, such as hospital-acquired pneumonia, can be better diagnosed with the assistance of precise methicillin-resistant Staphylococcus aureus (MRSA) testing. However, traditional methods necessitate high-tech tools, rigorous temperature cycling, and the extraction of genetic material from MRSA cells. Herein, we propose a sensitive, specific, and extraction-free strategy for MRSA detection by integrating allosteric probe-based target recognition and exonuclease-III (Exo-III)-enhanced color reaction. The penicillin-binding protein 2a (PBP2a) aptamer in the allosteric probe binds with MRSA to convert protein signals to nucleic acid signals. This is followed by the DNA polymerase-assisted target recycle and the production of numerous single-strand DNA (ssDNA) chains which bind with silver ion (Ag + ) aptamer to form a blunt terminus that can be identified by Exo-III. As a result, the Ag + aptamer pre-coupled to magnetic nanoparticles is digested. After magnetic separation, the Ag + in liquid supernatant catalyzes 3,3’,5,5’-tetramethylbenzidine (TMB) for a color reaction. In addition, a concentration of 54 cfu/mL is predicted to be the lowest detectable value. Based on this, our assay has a wide linear detection range, covering 5 orders of magnitude and demonstrating a high specificity, which allows it to accurately distinguish the target MRSA from other microorganisms.

CRISPR/Cas12a-Based APE1 Enzyme Cleavage Assay for Drug Resistance Analysis of Staphylococcus aureus-Related Pneumonia

“… 5 , 6 The sensitive and precise identification of MRSA is essential to preventing its detrimental effects and monitoring its spread within a population. Currently, MRSA detection is accomplished by the utilization of culture-based assays, 7 , 8 aptamer-mediated signal amplification tests, 9 , 10 and mass spectrometry-based approaches. 11 Nevertheless, these techniques typically necessitate a lengthy period for identification, significant manipulation of samples, and the use of costly apparatus.…”

“…aureus (MRSA) is a prevalent and highly drug-resistant bacterium that is responsible for generating significant mortality and morbidity in numerous diseases, , including pneumonia in child and hospital-acquired infections. , The sensitive and precise identification of MRSA is essential to preventing its detrimental effects and monitoring its spread within a population. Currently, MRSA detection is accomplished by the utilization of culture-based assays, , aptamer-mediated signal amplification tests, , and mass spectrometry-based approaches . Nevertheless, these techniques typically necessitate a lengthy period for identification, significant manipulation of samples, and the use of costly apparatus.…”