3种常见食源性致病菌多重重组酶聚合酶扩增检测方法研究

Detection of 3 kinds of common foodborne pathogenic bacteria by multiple recombinase polymerase amplification

中文关键词:  多重重组酶聚合酶扩增  沙门氏菌  单核细胞增生李斯特菌  蜡样芽孢杆菌  快速检测

英文关键词:multiple recombinase polymerase amplification  Salmonella  Listeria monocytogenes  Bacillus cereus  rapid detection

中文摘要:

      目的 建立食品中沙门氏菌、单核细胞增生李斯特菌和蜡样芽胞杆菌多重重组酶聚合酶扩增(recombinase polymerase amplification, RPA)快速检测方法。方法 选择沙门氏菌invA基因、单增李斯特菌hlyA基因和蜡样芽孢杆菌16S RNA序列为目标基因进行扩增, 建立并优化多重RPA扩增体系和扩增条件; 评价反应体系的特异性和灵敏度, 并对人工污染食品样品和实际样品进行检测。结果 多重RPA反应体系能够在 20 min完成3种目标基因的扩增, 特异性强; 对沙门氏菌、单增李斯特菌和蜡样芽孢杆菌的灵敏度分别为2.70×105、1.30×105、1.44×104 CFU/mL, 能够用于人工污染样品和实际样品的检测。结论 本研究建立的多重RPA等温扩增方法特异性强, 操作快速、简单, 可为食源性致病菌的快速检测提供新方向。

英文摘要:

      Objective To establish a rapid detection method for Salmonella, Listeria monocytogenes and Bacillus cereus by multiple recombinase polymerase amplification (RPA). Methods Selecting Salmonella invA gene, Listeria monocytogenes hlyA gene and Bacillus cereus 16S RNA sequence as target genes for amplification, and establishing and optimizing a multiple RPA amplification system and amplification conditions; the specificities and sensitivities of the reaction systems were evaluated and artificially contaminated food samples and actual samples were tested. Results The multiple RPA reaction system was able to complete the amplification of the 3 kinds of target genes in 20 min, with strong specificity; the sensitivities to Salmonella, Listeria monocytogenes and Bacillus cereus were 2.70×105, 1.30×105, 1.44×104 CFU/mL, respectively, and could be used for detecting artificially contaminated samples and actual samples. Conclusion The multiple RPA isothermal amplification method established in this study is specific, rapid and simple, and can provide a new direction for rapid detection of foodborne pathogenic bacteria.