猪肉中常见致病菌的多重直接PCR法的建立与应用

发布时间：2019-07-01 19:26

【摘要】：目的建立检测大肠杆菌、沙门氏菌、金黄色葡萄球菌、李斯特菌和小肠耶尔森氏菌5种致病菌的多重直接PCR方法。方法根据大肠杆菌phoA、沙门氏菌invA、金黄色葡萄球菌nuc、李斯特菌hly、小肠耶尔森氏菌ail的基因序列,设计多重直接PCR引物,建立并优化多重直接PCR反应条件,检测引物的扩增特异性和灵敏性,并将所建立的方法应用于实际采集猪肉样本的检测,与金标培养法相比较,计算多重直接PCR检测法的敏感性、准确性以及阳性预测值。结果所设计的多重直接PCR引物对5种菌都有特异扩增,最低检出限量大肠杆菌为10CFU,金黄色葡萄球菌敏感性为100CFU,沙门氏菌、李斯特菌、小肠耶尔森氏菌敏感性可达1CFU。60份猪肉样本中检出大肠杆菌15份、沙门氏菌6份、金黄色葡萄球菌21份、李斯特菌20份、小肠耶尔森氏菌35份,阳性检出率均高于金标培养法,总体检测敏感性为100%,准确性为94%,阳性预测值为81.44%。结论多重直接PCR法实现了同时对各食源性致病菌敏感特异的检测,并且省去了提取模板的步骤,将检测时间缩短至3h左右,便于食品安全风险监测中常见食源性致病菌的通量检测。
[Abstract]:Objective to establish a multiplex direct PCR method for the detection of E. coli, Salmonella, Staphylococcus aureus, Listeria monocytogenes and Yelseng small intestine. Methods according to the gene sequence of E. coli phoA, Salmonella aureus invA, Staphylococcus aureus nuc, hly, ail, multiple direct PCR primers were designed, the multiplex direct PCR reaction conditions were established and optimized, and the amplification specificity and sensitivity of the primers were detected. The established method was applied to the detection of pork samples and compared with the gold standard culture method. The sensitivity, accuracy and positive predictive value of multiple direct PCR detection were calculated. Results the designed multiplex direct PCR primers were specifically amplified to all five kinds of bacteria. The minimum detection limit of Escherichia coli was 10 CFU, Staphylococcus aureus sensitivity was 100 CFU, salmonella, Listeria monocytogenes, Yelseng small intestine were as sensitive as 15 E. coli, 6 Salmonella, 21 Staphylococcus aureus, 20 Listeria monocytogenes and 35 Yelseng small intestine samples, among which 15 were Escherichia coli, 6 were Salmonella, 21 were Staphylococcus aureus, 20 were Listeria, 35 were Yelseng, and 15 were E. coli, 6 were Salmonella, 21 were Staphylococcus aureus, 21 were Staphylococcus aureus, 20 were Staphylococcus aureus, and 35 were Yelsonia. The positive detection rate was higher than that of gold standard culture method. The overall sensitivity, accuracy and positive predictive value were 100%, 94% and 81.44%, respectively. Conclusion multiple direct PCR method realizes the sensitive and specific detection of food-borne pathogenic bacteria at the same time, and saves the step of extracting template, and shortens the detection time to about 3 hours, which is convenient for the flux detection of common food-borne pathogenic bacteria in food safety risk monitoring.
【作者单位】：中国动物卫生与流行病学中心农业部畜禽产品质量安全风险评估实验室(青岛);吉林农业大学生命科学学院;
【基金】：农业部“国家农产品质量安全风险评估重大专项”(No.GJFP2016007)资助留学人员科技活动项目择优资助~~
【分类号】：R155.5

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