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Establishment and Application of an Assay for Detecting Staphylococcus aureus Using Asymmetric Recombinase-Mediated Amplification Combined with Molecular Beacons—Third Military Medical University
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Objective: To establish a rapid, isothermal detection method for pathogenic bacteria based on the combination of recombinase-mediated amplification (RAA) technology and molecular beacons. Methodology: The method was developed by designing specific primers and molecular beacon probes targeting the gene encoding Staphylococcus aureus protein A (SPA). The primer concentration ratio was systematically adjusted to determine the optimal primer concentration ratio for asymmetric amplification. Asymmetric recombinase-mediated amplification was then performed, followed by hybridization with molecular beacon probes. The results were analyzed by agarose gel electrophoresis and fluorescence detection. The sensitivity of the method was evaluated by serially diluting a positive plasmid at 10-fold increments. To assess the specificity of the RAA hybridization assay, 72 strains—including Staphylococcus aureus and other species of the genus Staphylococcus—that had been preserved in the Microbiology Laboratory of the Daping Hospital’s Department of Laboratory Medicine since December 2016 were tested using this method. Based on the specificity study, an additional 39 strains—also preserved in our laboratory and collected in December 2016—were included to perform Kappa consistency analysis and evaluate the clinical diagnostic performance of the assay.
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