单增李斯特菌RAA-exo检测方法的建立和应用

Development and application of a recombinase-aided amplification-exonuclease assay for detecting Listeria monocytogenes

摘要:

单增李斯特菌(Listeria monocytogenes, LM)是引起食源性疾病暴发的主要病原之一，严重威胁食品安全和公共卫生安全。在食源性致病菌污染呈现多元化特征的环境下，特异性检测单增李斯特菌并开展预防工作极为重要。目的 建立一种新的单增李斯特菌重组酶辅助扩增-外切酶(recombinase aided amplification-exonuclease, RAA-exo)检测方法。方法 针对单增李斯特菌的hly基因设计多对RAA引物和探针，筛选核酸扩增效率最高的引物。利用最佳RAA引物系统对反应体系进行优化，包括A buffer、B buffer、RAA引物和探针的使用量。基于优化后的反应体系，对该方法的灵敏度、特异性和实际应用能力进行评估。结果 本方法检测重组质粒的最低检测限为0.5 copies/μL，检测单增李斯特菌液的最低检测限为10 CFU/mL。该方法仅对靶细菌单增李斯特菌敏感，对沙门氏菌、大肠杆菌、金黄色葡萄球菌、蜡样芽孢杆菌等几种常见食源性病原菌均无法检出，对绵羊李斯特菌、西尔李斯特菌、英诺克李斯特菌等几种李斯特菌同样无法检出。对44份猪肉样本的应用结果表明，本方法的检测结果与行业标准SN/T 5224—2019推荐的实时荧光PCR内标法结果一致。结论 本研究建立的RAA-exo技术具有灵敏度高、特异性强、操作简便等优点，在37 ℃条件下20 min内即可完成检测，适用于基层现场快速检测，为单增李斯特菌的快速检测提供了高效、便捷的技术支持，具有广阔的应用前景。

Abstract:

Listeria monocytogenes, as a major causative agent of foodborne illness outbreaks, poses a serious threat to food safety and public health. In complex foodborne pathogen environments, the specific and effective detection methods for L. monocytogenes are crucial.Objective To develop a novel recombinase-aided amplification-exonuclease (RAA-exo) assay for detecting L. monocytogenes.Methods Multiple RAA primer and probe sets targeting hly were designed, and the optimal primer set was selected based on nucleic acid amplification efficiency. The reaction system was rigorously optimized, focusing on the concentrations of A buffer, B buffer, and RAA primers and probe.Results The optimized RAA-exo assay showed a limit of detection (LOD) of 0.5 copies/μL for recombinant plasmids and 10 CFU/mL for L. monocytogenes suspensions. The assay demonstrated high specificity, selectively detecting L. monocytogenes without cross-reactivity to other common foodborne pathogens, including Salmonella, Escherichia coli, Staphylococcus aureus, Bacillus cereus, or other Listeria species (L. ovinae, L. seeligeri, and L. innocua). In a validation study using 44 pork samples, the RAA-exo assay results were in complete agreement with those of the real-time fluorescence PCR internal standard method outlined in the industry standard SN/T 5224—2019.Conclusion The developed RAA-exo assay exhibits high sensitivity and specificity, requires minimal hands-on time, and achieves detection within 20 min at 37 °C. Therefore, the assay is suitable for rapid, on-site testing, serving as an efficient and convenient tool for L. monocytogenes detection, with promising applications in food safety monitoring.