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A Study on the Rapid Detection of Staphylococcus aureus Using CRISPR-Cas13a-Assisted RAA—Nankai & Jiangsu CDC.pdf
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【Abstract】 Objective: To establish a rapid molecular detection technique for Staphylococcus aureus. Methods: Specific primers were designed and synthesized based on the conserved region of the thermostable nuclease gene (nuc) in Staphylococcus aureus. By optimizing reaction conditions, a recombinase-mediated isothermal amplification (RAA) assay for Staphylococcus aureus was developed. The CRISPR-Cas13a protein was expressed and purified, and specific cRNAs (CRISPR RNAs) were designed. The crRNA-guided CRISPR-Cas13a protein was used to detect RAA products. The optimized method was evaluated for sensitivity and specificity. Furthermore, this method was compared with real-time PCR for the detection of Staphylococcus aureus in food samples, and the agreement between the two methods was assessed. Results: The CRISPR-Cas13a-assisted RAA assay demonstrated a detection sensitivity of 10 CFU/ml for Staphylococcus aureus, which was comparable to that of real-time PCR at approximately 10 CFU/ml. The assay took only 30 minutes to complete. No cross-reactivity was observed with other foodborne pathogens. The positive rates obtained by both the CRISPR-Cas13a-assisted RAA assay and real-time PCR were 8.75% when testing 80 food samples, showing high agreement (Kappa = 1, P > 0.05). Conclusion: The established CRISPR-Cas13a-assisted RAA method offers advantages of simplicity, rapidity, high sensitivity, and specificity, providing a novel technical approach for the detection of Staphylococcus aureus.
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