大肠埃希菌O157:H7的实时荧光PCR检测方法研究

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。

Study on Real - time PCR Detection Methods for Escherichia coli O157:H7

Objective To establish a sensitive,specific,and rapid method for detection of Escherichia coli O157: H7,and to apply it in the emergent public health events and epidemiological investigation of foodborne pathogenic bacteria. Methods According to the GenBank Escherichia coli O157: H7rfbE gene sequence, the primers and TaqMan probe were designed.The real-time fluorescent PCR reaction conditions were optimized.The real-time fluorescent PCR detection system for Escherichia coli O157: H7 was established.And the specificity,sensitivity and reproducibility of the method were evaluated. Results The test results of Escherichia coli O157: H7 strains were positive,whereas the test results of all other strains were negative.The sensitivity of the detection method was up to 1×102 cfu/ml.The simulated samples of pork,mutton,chicken,fresh vegetables were found to be contaminated with 1×104cfu/mL bacteria.The duration between nucleic acid extraction to bacteria detection was approximately 3 hours. Conclusions The established real-time fluorescence PCR detection method has the advantages of high sensitivity,strong specificity and rapidness,which can be used in Escherichia coli O157: H7 food poisoning diagnosis and a rapid microbiological testing of food.It provides a new detection way for the molecular epidemiological survey of foodborne diseases.