多重重组酶介导等温扩增技术检测食品中3种食源性致病菌

Determination of 3 kinds of foodborne pathogens in food by multiplex recombinase aided amplification technology

中文关键词:  重组酶介导扩增技术  大肠埃希氏菌O157:H7  沙门氏菌  金黄色葡萄球菌

英文关键词:recombinase aided amplification  Escherichia coli O157:H7  Salmonella  Staphylococcus aureus

中文摘要:

      目的 建立快速、灵敏的多重重组酶介导等温扩增法(recombinase aided amplification, RAA)同时检测食品中的大肠埃希氏菌O157:H7、沙门氏菌和金黄色葡萄球菌。方法 根据大肠埃希氏菌O157:H7的rfbE基因、沙门氏菌的invA基因以及金黄色葡萄球菌的nuc基因的序列保守区域设计RAA特异性引物, 筛选出最优的引物组合并优化各项实验条件; 通过标准菌株验证方法特异性和灵敏度, 并通过人工污染实验, 验证方法在实际食品样本中的检测能力。结果 多重RAA方法最佳扩增温度为37℃, 反应时间为50 min; 方法特异性良好, 仅对目标菌株存在特异性扩增条带, 与其他常见食源性致病菌无交叉反应; 方法对基因组DNA的检测灵敏度为0.10 ng/μL; 对经过简单增菌的人工污染牛奶样本和牛肉干样本中目标菌株的检出限为100 CFU/mL。结论 本研究建立的多重RAA扩增方法具有特异性好、灵敏度高、检测通量高等优势, 适用于多种场景下食品样本中3种食源性致病菌的快速检测。

英文摘要:

      Objective To establish a rapid and sensitive multiplex recombinase aided amplification (RAA) method for simultaneous detection of Escherichia coli O157:H7, Salmonella and Staphylococcus aureus in food. Methods RAA specific primers were designed based on the sequence conserved regions of the rfbE gene of Escherichia coli O157:H7, the invA gene of Salmonella and the nuc gene of Staphylococcus aureus. The optimal primer combination was screened and various experimental conditions were optimized. The specificity and sensitivity of the method were verified through standard strains, and the detection ability of the method in actual food samples was verified through artificial contamination experiments. Results The optimal amplification temperature of the multiple RAA method was 37℃ and the reaction time was 50 min. The method was very specific, with only specific amplification bands for the target strains and no cross reaction with other common foodborne pathogens. The sensitivity of the method for genomic DNA was 0.10 ng/μL. The limit of detection of the target strain in artificially contaminated milk samples and beef jerky samples after simple enrichment was 100 CFU/mL. Conclusion The multiple RAA amplification method established in this study has the advantages of good specificity, high sensitivity, and high detection throughput, and is suitable for the rapid detection of 3 kinds of foodborne pathogens in food samples in various scenarios.