Abstract
The rapid emergence of antimicrobial resistant bacteria requires the development of new diagnostic tests. Nucleic acid-based assays determine antimicrobial susceptibility by detecting genes that encode for the resistance. In this study, we demonstrate rapid and simultaneous detection of three genes that confer resistance in bacteria to extended spectrum β-lactam and carbapenem antibiotics; CTX-M-15, KPC and NDM-1. The assay uses isothermal DNA amplification (recombinase polymerase amplification, RPA) implemented on a programmable digital microfluidics (DMF) platform. Automated dispensing protocols are used to simultaneously manipulate 45 droplets of nL volume containing sample DNA, reagents, and controls. The droplets are processed and mixed under electronic control on the DMF devices with positive amplification measured by fluorescence. The assay on these devices is significantly improved with a Time to Positivity (TTP) half that of the benchtop assay.
Highlights
The emergence of antibiotic resistant bacteria is considered one of the greatest threats to human health as infections are becoming increasingly common [1]
The sample was continuously pumped across the surface of an 8.3 μL reaction chamber, and they were able to amplify specific target sequences from three pathogens Neisseria gonorrhoeae, Salmonella enterica, and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA [26]
The triplex assay identifies and can quantify three of the key genes responsible for antimicrobial resistance in Gram-negative microorganisms in a mixed sample droplet in a short time window. These genes are New Delhi metallo-β-lactamase (NDM)-1, Klebsiella pneumoniae carbapenemase (KPC), and CTX-M-15 that encode for carbapenemases and an ESBL respectively
Summary
The emergence of antibiotic resistant bacteria is considered one of the greatest threats to human health as infections are becoming increasingly common [1]. The sample was continuously pumped across the surface of an 8.3 μL reaction chamber, and they were able to amplify specific target sequences from three pathogens Neisseria gonorrhoeae, Salmonella enterica, and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA [26] They demonstrated detection in 20 min and a LoD of extracted DNA from between 10 and 100 cfu. The triplex assay identifies and can quantify three of the key genes responsible for antimicrobial resistance in Gram-negative microorganisms in a mixed sample droplet in a short time window These genes are NDM-1, KPC, and CTX-M-15 that encode for carbapenemases and an ESBL respectively. The genes were identified using isothermal amplification methods and a fluorescent readout
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