A Programmable Digital Microfluidic Assay for the Simultaneous Detection of Multiple Anti-Microbial Resistance Genes

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 beta-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.