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Focusing on the research and development and application of isothermal nucleic acid amplification technology
不对称重组酶介导扩增结合分子信标检测金黄色葡萄球菌方法的建立与应用-第三军医大
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Objective: To establish a rapid, isothermal detection method for pathogenic bacteria based on recombinase-mediated amplification (RAA) combined with molecular beacons. Methods: The methodology was developed by designing specific primers and molecular beacon probes targeting the gene encoding Staphylococcus aureus protein A (SPA). The optimal primer concentration ratio was determined through systematic adjustments in primer ratios during asymmetric PCR amplification. Subsequently, the asymmetrically amplified products were hybridized with the molecular beacon probes, and the results were visualized using agarose gel electrophoresis followed by fluorescence detection. To evaluate the sensitivity of the method, a positive plasmid was serially diluted in 10-fold increments. Additionally, the specificity of the RAA-hybridization assay was assessed by testing 72 bacterial strains—comprising Staphylococcus aureus and other species within the genus Staphylococcus—that had been preserved at the Microbiology Laboratory of the Daping Hospital’s Department of Clinical Testing since December 2016. Finally, based on the specificity findings, 39 additional bacterial strains isolated in December 2016 and stored in our laboratory were included to perform Kappa consistency analysis and evaluate the clinical diagnostic performance of the method.
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