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Focusing on the research and development and application of isothermal nucleic acid amplification technology
RAA联合CRISPR-Cas13a快速检测4种腹泻病原菌-安柏霖.pdf
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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). Primer concentrations were systematically adjusted to identify the optimal primer ratio for asymmetric amplification. Asymmetric RAA was then performed, followed by hybridization with molecular beacon probes. Results were visualized using agarose gel electrophoresis and fluorescence detection. To evaluate the method's sensitivity, positive plasmids were serially diluted in 10-fold increments. Additionally, the specificity of the assay was assessed by testing 72 bacterial strains—collected in December 2016 from the Microbiology Laboratory at Daping Hospital—including both Staphylococcus aureus and other species within the genus Staphylococcus. On the basis of these specificity tests, 39 additional bacterial strains preserved in our laboratory (also collected in December 2016) were included to conduct a Kappa consistency analysis and evaluate the clinical diagnostic performance of the method. Results: The highest efficiency in generating single-stranded DNA (sDNA) was achieved when the concentration ratio of restrictive to non-restrictive primers was set at 1:20. Moreover, asymmetric RAA exhibited significantly higher hybridization efficiency compared to symmetric amplification. The method demonstrated a detection limit as low as 20 copies per reaction. Importantly, this RAA-based hybridization assay successfully distinguished Staphylococcus aureus from other species within the genus Staphylococcus, showing excellent agreement with the traditional gold standard, as indicated by a Kappa value of 0.860. When applied to a panel of 111 bacterial strains, the assay achieved a sensitivity of 92.5% (37 out of 40), a specificity of 97.2% (69 out of 71), a positive predictive value of 94.9% (37 out of 39), and a negative predictive value of 95.8% (69 out of 72). The positive likelihood ratio was 33.04, while the negative likelihood ratio was 0.077. The resulting Youden index stood at 0.897. Furthermore, when compared with the conventional gold standard, the RAA hybridization assay showed nearly perfect agreement, with a Kappa value of 0.902. Conclusion: By optimizing the conditions for asymmetric RAA and integrating it with molecular beacon probes, we have established a novel RAA-based hybridization technique for detecting bacterial DNA. This approach lays a solid foundation for applying isothermal amplification methods in clinical diagnostics.
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