Integration of Nucleic Acid Amplification, Detection, and Melting Curve Analysis for Rapid Genotyping of Antimicrobial Resistance

To prevent the overuse of antibiotics and reduce the spread of antimicrobial resistance (AMR), we developed an integrated genotypic test to identify target AMR genes and mutations with minimal user operation. The integrated AMR sensor consists of a reaction chamber built on a total internal reflection (TIR)-coupled DNA microarray, a temperature management unit, and a compact fluorescence reader. By executing a programmed temperature profile, the automated system can perform asymmetric polymerase chain reactions (PCR) to amplify multiple target genes, microarray hybridizations to detect the amplicons, and melting curve analysis (MCA) to identify resistance mutations. Eight AMR genes selected from Acinetobacter baumannii, Klebsiella pneumonia, Escherichia coli, Campylobacter coli, and Campylobacter jejuni were amplified using the asymmetric PCR and subsequently detected using the TIR-coupled microarray. The point mutation in the quinolone resistance-determining region of the gyrA gene of Campylobacter jejuni was further studied by performing MCA on the TIR-coupled microarray. The benefits of integrated and rapid assay, compact and automated instrument, and multiplexed analysis would facilitate point-of-care antimicrobial susceptibility testing for bacterial infections.