数字PCR定量检测食品中单核细胞增生李斯特氏菌方法的研究

Detection of Listeria monocytogenes in foods by droplet digital PCR

中文关键词:  单核细胞增生李斯特氏菌  微滴式数字PCR  定量

英文关键词:Listeria monocytogenes  droplet digital PCR  quantitation

中文摘要:

      目的 建立微滴式数字PCR技术(droplet digital PCR, ddPCR)定量检测食品中单核细胞增生李斯特氏菌的分析方法。方法 选择基因hly A为靶序列, 设计PCR扩增引物和TaqMan探针, 优化反应条件和反应体系, 通过细菌分离和ddPCR方法对靶标基因的检测特异性、灵敏度和重复性进行实验, 并对定量结果进行分析。结果 ddPCR反应中的最佳探针浓度为5 pmol/μL, 特异性好, 检出限为(3.6?0.1) copies/20 μL, 重复性实验良好, 标准偏差为0.067%。ddPCR的拷贝数(copy number)与细菌密度(colony forming units, CFU/mL)形成了较好的线性关系。结论 本研究建立ddPCR的拷贝数和菌液密度或菌落数的线性关系, 可以为单核细胞增生李斯特氏菌的快速定量检测提供参考。

英文摘要:

      Objective To establish a new quantitative accuracy method for detection of Listeria monocytogenes by droplet digital PCR (ddPCR) in food. Methods A droplet digital polymerase chain reaction (ddPCR) method for quantifying L. monocytogenes based on hly A gene was developed, and the primer and probe concentration in ddPCR was optimized. Specificity, sensitivity and repeatability were tested based on ddPCR and quantitative accuracy analysis was performed on the results. Results The optimized probe concentration was 5 pmol/μL. The minimum DNA copies of Listeria monocytogenes was (3.6?0.1) copies/20 μL by ddPCR, with high specificity, and the repeatability test was good and the standard deviation was 0.067%. A good linear relation was established between the copy number by ddPCR and the bacterial colony forming units (CFU) of Listeria monocytogenes. Conclusion The linear relationship between the copy numbers of ddPCR and the bacterial density can be used as a reference for the rapid quantitative detection of Listeria monocytogenes.