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Focusing on the research and development and application of isothermal nucleic acid amplification technology
CRISPR_Cas13a辅助RAA快速检测金黄色葡萄球菌的研究-南开&江苏疾控
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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 sequence of the thermostable nuclease gene (nuc) in S. aureus. By optimizing reaction conditions, a recombinase-aided amplification (RAA) assay was developed for the sensitive and specific detection of S. aureus. Additionally, the CRISPR-Cas13a protein was expressed and purified, and a specific cIRNA (CRISPR RNA) was designed to guide Cas13a in detecting RAA products. The optimized method was evaluated for its sensitivity and specificity, and then applied alongside real-time PCR to detect S. aureus in food samples, with consistency assessed by comparing the results from both methods. Results: The CRISPR-Cas13a-assisted RAA assay demonstrated a detection limit of 10 CFU/ml for S. aureus, which is comparable to real-time PCR but significantly faster—requiring only 30 minutes. Importantly, no cross-reactivity was observed with other foodborne pathogens. Furthermore, when applied to 80 food samples, both the CRISPR-Cas13a-assisted RAA method and real-time PCR yielded identical positive rates of 8.75%, indicating high agreement (Kappa = 1, P > 0.05). Conclusion: The newly established CRISPR-Cas13a-assisted RAA method offers several advantages, including simplicity, speed, high sensitivity, and excellent specificity, providing a novel and effective tool for the detection of S. aureus.
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