摘要
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 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.
出处
《实用预防医学》
CAS
2012年第3期362-364,共3页
Practical Preventive Medicine
基金
宝鸡市卫生局科研立项课题(2011-45)
