5种食源性致病菌PCR检测方法的建立

Establishment of PCR method for detecting 5 kinds of food-borne pathogenic bacteria

中文关键词:  沙门氏菌  副溶血性弧菌  大肠埃希氏菌O157:H7  金黄色葡萄球菌  单核细胞增生李斯特氏菌  聚合酶链式反应

英文关键词:Salmonella  Vibrio parahemolyticus  Escherichia coli O157:H7  Staphylococcus aureus  Listeria monocytogenes  polymerase chain reaction

中文摘要:

      目的 建立食品中的沙门氏菌、副溶血性弧菌、大肠埃希氏菌O157:H7、金黄色葡萄球菌和单核细胞增生李斯特氏菌的PCR快速检测方法。方法 以缓冲蛋白胨水为增菌培养基, 采用直接提取法提取DNA并测定其核酸浓度。对PCR的退火温度、模板浓度进行优化, 把5种目标菌分为两组进行检测分析, 并进行特异性和灵敏度实验。结果 5种目标菌培养后均表现出良好的生长趋势。PCR扩增最佳退火温度为59 ℃, 5种目标菌的引物特异性好, 方法的检测灵敏度高。沙门氏菌、副溶血性弧菌、大肠埃希氏菌O157:H7、金黄色葡萄球菌和单核细胞增生李斯特氏菌最低检出核酸浓度分别为0.0202、0.158、0.187、2.30和1.05 μg/mL。结论 该方法简便、快速、灵敏度高, 能满足食品安全检测要求。

英文摘要:

      Objective To establish a rapid method for the simultaneous detection of Salmonella, Vibrio parahemolyticus, Escherichia coli O157:H7, Staphylococcus aureus and Listeria monocytogenes by PCR method. Methods Buffered peptone water was used as enrichment medium, the DNA was extracted by direct extraction method and the concentration of DNA nucleic acid was determined. The annealing temperature and template concentration of PCR were optimized. The 5 kinds of target bacteria were divided into 2 groups for detection and analysis, and their specificity and sensitivity were tested. Results All the 5 kinds of target bacteria showed good growth trend after culture. The best annealing temperature for this test was 59 ℃, and the detection of 5 kinds of target bacteria had good specificity and high sensitivity. The lowest concentrations of nucleic acid detected of Salmonella, Vibrio parahemolyticus, Escherichia coli O157:H7, Staphylococcus aureus and Listeria monocytogenes were 0.0202, 0.158, 0.187, 2.30 and 1.05 μg/mL, respectively. Conclusion This method is simple, fast and sensitive, which can meet the requirements of food safety testing.