Rapid and Sensitive Detection of Antibiotic Resistance Genes by Utilizing TALEs as a Diagnostic Probe with 2D-Nanosheet Graphene Oxide

As antibiotic resistance has risen as one of the majorhealth concernsassociated with infectious diseases due to the reduced efficacy ofantibiotics, rapid and sensitive detection of antibiotic resistancegenes is critical for more effective and faster treatment of infectiousdiseases. A class of programmable DNA-binding domains called transcriptionalactivator-like effectors (TALEs) provides a novel scaffold for designingversatile DNA-binding proteins due to their modularity and predictability.Here, we developed a simple, rapid, and sensitive system for detectingantibiotic resistance genes by exploring the potential of TALE proteinsfor the creation of a sequence-specific DNA diagnostic along with2D-nanosheet graphene oxide (GO). TALEs were engineered to directlyrecognize the specific double-stranded (ds) DNA sequences presentin the tetracycline resistance gene (tetM), avoidingthe need for dsDNA denaturation and renaturation. We take advantageof the GO as an effective signal quencher to quantum dot (QD)-labeledTALEs for creating a turn-on strategy. QD-labeled TALEs are adsorbedon the GO surface, which will bring QDs in close proximity to GO.Due to the fluorescence quenching ability of GO, QDs are expectedto be quenched by GO via fluorescence resonance energy transfer (FRET).QD-labeled TALE binding to the target dsDNA would lead to the conformationalchange, which would result in dissociation from the GO surface, therebyrestoring the fluorescence signal. Our sensing system was able todetect low concentrations of dsDNA sequences in the tetM gene after only 10-minute incubation with the DNA, providing a limitof detection as low as 1 fM of Staphylococcus aureus genomic DNA. This study demonstrated that our approach of utilizingTALEs as a new diagnostic probe along with GO as a sensing platformcan provide a highly sensitive and rapid method for direct detectionof the antibiotic resistance gene without requiring DNA amplificationor labeling.

Automated summary

Antibiotic resistance has become a major health concern due to the reduced efficacy of antibiotics. This study developed a rapid and sensitive system for detecting antibiotic resistance genes using programmable DNA-binding domains called TALEs and graphene oxide (GO). The results showed that the combination of TALEs and GO can provide a highly sensitive and rapid method for direct detection of antibiotic resistance genes.