重组酶介导等温扩增法检测食品中铜绿假单胞菌

Detection of Pseudomonas aeruginosa in food by recombinase-aided amplification method

中文关键词:  铜绿假单胞菌  lasB基因  重组酶介导等温扩增

英文关键词:Pseudomonas aeruginosa  lasB gene  recombinase-aided amplification

中文摘要:

      目的 建立重组酶介导等温扩增(recombinase-aided amplification, RAA)法快速、灵敏、特异检测食品中铜绿假单胞菌的分析方法。方法 针对铜绿假单胞菌lasB基因的特异性保守区域, 设计RAA特异性引物和exo探针, 筛选出最优组合; 对提取的目标基因组DNA进行检测, 确定方法的特异性和灵敏度, 并通过人工污染实验, 比对不同检测方法对食品样品的检出限。结果 建立的铜绿假单胞菌lasB基因RAA检测方法特异性良好, 仅对铜绿假单胞菌有扩增曲线, 对其他致病菌无交叉反应; 方法对纯菌液的灵敏度为1.7 pg/μL。人工污染实验表明, 方法对肉类样品中铜绿假单胞菌的检出限与传统方法一致, 达1.0×103 CFU/mL; RAA方法检测时间仅需20 min。结论 本研究建立的铜绿假单胞菌实时荧光RAA检测方法特异性强、灵敏度高、反应时间短, 可用于食品中铜绿假单胞菌的快速检测。

英文摘要:

      Objective To develop a rapid, sensitive and specific method for the detection of Pseudomonas aeruginosa in food by recombinase-aided amplification (RAA). Methods RAA specific primers and exo probes were designed according to the specific conserved region of lasB gene of Pseudomonas aeruginosa, and selected the optimal combination. The extracted target genomic DNA was tested to determine the specificity and sensitivity of the method. The limits of detection of different detection methods for food samples were compared through artificial contamination experiments. Results The established RAA method for detecting the lasB gene of Pseudomonas aeruginosa had good specificity, and only had an amplification curve for Pseudomonas aeruginosa, but no cross-reaction for other pathogens. The sensitivity of the method to pure bacterial solution was 1.7 pg/μL. The artificial contamination experiment showed that the limit of detection of Pseudomonas aeruginosa in meat samples was 1.0×103 CFU/mL, which was consistent with the traditional methods. The detection time of RAA method was only 20 min. Conclusion The real-time fluorescent RAA detection method of Pseudomonas aeruginosa established in this study has strong specificity, high sensitivity and short reaction time, which can be used for the rapid detection of Pseudomonas aeruginosa in food.