| Summary: | The rise of multidrug resistance of <i>Pseudomonas aeruginosa</i> highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including <i>P</i>. <i>aeruginosa</i>. Detection of efflux genes using a polymerase chain reaction (PCR)-based system would enable resistance detection and aid clinical decision making. Therefore, we aimed to develop and optimize a novel method herein referred to as “<i>effluxR</i> detection assay” using multiplex digital PCR (mdPCR) for detection of <i>mex</i> efflux pump genes in <i>P. aeruginosa</i> strains. The annealing/extension temperatures and gDNA concentrations were optimized to amplify <i>mexB</i>, <i>mexD</i>, and <i>mexY</i> using the multiplex quantitative PCR (mqPCR) system. We established the optimal mqPCR conditions for the assay (Ta of 59 °C with gDNA concentrations at or above 0.5 ng/µL). Using these conditions, we were able to successfully detect the presence of these genes in a quantity-dependent manner. The limit of detection for <i>mex</i> genes using the <i>effluxR</i> detection assay with mdPCR was 0.001 ng/µL (7.04–34.81 copies/µL). Moreover, using blind sample testing, we show that <i>effluxR</i> detection assay had 100% sensitivity and specificity for detecting <i>mex</i> genes in <i>P</i>. <i>aeruginosa</i>. In conclusion, the <i>effluxR</i> detection assay, using mdPCR, is able to identify the presence of multiple <i>mex</i> genes in <i>P</i>. <i>aeruginosa</i> that may aid clinical laboratory decisions and further epidemiological studies.
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