Abstract
The increasing emergence of multidrug- and pan-resistant pathogens requires rapid and cost-efficient diagnostic tools to contain their further spread in healthcare facilities and the environment. The currently established diagnostic technologies are of limited utility for efficient infection control measures because they are either cultivation-based and time-consuming or require sophisticated assays that are expensive. Furthermore, infectious diseases are unfortunately most problematic in countries with low-resource settings in their healthcare systems. In this study, we developed a cost-efficient detection technology that uses G-quadruplex DNAzymes to convert a chromogenic substrate resulting in a color change in the presence of antibiotic resistance genes. The assay is based on padlock probes capable of high-multiplex reactions and targets 27 clinically relevant antibiotic resistance genes associated with sepsis. In addition to an experimental proof-of-principle using synthetic target DNA, the assay was evaluated with multidrug-resistant clinical isolates.
Highlights
The emerging antibiotic resistance crisis represents a serious healthcare problem and was declared by the WHO as one of the biggest threats to global health, food security, and development
We have demonstrated the successful proof-of-principle that G-quadruplexbased DNAzymes can be used in combination with padlock probes making a colorimetric readout in the presence of a target gene possible
Future optimizations that have a strong impact on the specificity of the reaction must include the padlock probe design and the rolling circle amplification (RCA)
Summary
The emerging antibiotic resistance crisis represents a serious healthcare problem and was declared by the WHO as one of the biggest threats to global health, food security, and development. Novel technologies that are significantly more cost-effective and able to detect hundreds of antibiotic resistance genes in parallel have still to be developed. Single-stranded oligonucleotides with two terminal recognition regions complementary to a target, a primer attachment site, and usually a DNA barcode sequence (Figure 1) [1]. We integrated a complementary G-quadruplex-hemin DNAzyme sequence into the padlock probe. This sequence is amplified during an RCA and quence into the padlock probe. This sequence is amplified during an RCA and monomerized using a restriction site integrated into the primer attachment site. Interest, a primer binding site with a restriction site, and a G-quadruplex sequence
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