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目的建立一种敏感、特异、快速的大肠埃希菌O157:H7的检测方法,应用于突发公共卫生事件及食源性致病菌流行病学调查的检测。方法根据GenBank大肠埃希菌O157:H7rfbE基因序列,设计引物和TaqMan探针,对实时荧光PCR反应条件进行优化,建立实时荧光PCR检测大肠埃希菌O157:H7的反应体系,并对该法的特异性、敏感性和重复性进行评价。结果大肠埃希菌O157:H7菌株的检测结果均为阳性,而所有其它菌株检测结果均为阴性;该方法检测的灵敏度可达1×102cfu/ml。模拟污染的猪肉、羊肉、鸡肉、生食蔬菜样品,均可检出1×104cfu/ml的细菌。从细菌核酸提取至完成检测约需3 h。结论建立的实时荧光PCR检测方法具有灵敏度高、特异性强、快速等优点,可用于大肠埃希菌O157:H7食物中毒的快速诊断和食品微生物检测,为食源性疾病的分子流行病学调查提供新的检测手段。
Objective To establish a sensitive, specific and rapid method for the detection of Escherichia coli O157: H7 in public health emergencies and epidemiological investigation of foodborne pathogens. Methods According to the gene sequence of Escherichia coli O157: H7rfbE in GenBank, primers and TaqMan probes were designed to optimize real-time PCR reaction conditions. Real-time PCR was used to detect the reaction system of Escherichia coli O157: H7. Specificity, sensitivity and reproducibility. Results The test results of Escherichia coli O157: H7 were all positive and the results of all the other strains were negative. The sensitivity of this method was 1 × 102cfu / ml. Simulated contaminated pork, lamb, chicken, raw vegetables samples, can detect 1 × 104cfu / ml of bacteria. From bacterial nucleic acid extraction to completion of the test takes about 3 h. Conclusion The established real-time fluorescence PCR assay has the advantages of high sensitivity, specificity, rapidity and so on. It can be used in the rapid diagnosis of food poisoning caused by Escherichia coli O157: H7 and the detection of food micro-organisms. It is a molecular epidemiological investigation of foodborne diseases Provide new testing methods.