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目的建立免疫磁珠富集(immunomagnetic separation,IMS)-实时荧光PCR(q PCR)技术快速检测肠出血性大肠埃希菌O157∶H7的方法。方法根据肠出血性大肠埃希菌O157∶H7抗原特异性基因rfb EO157设计引物和Taq Man探针,建立q PCR检测体系;对样品中的O157∶H7大肠埃希菌用出血性大肠O157抗体标记免疫磁珠进行特异性捕获,再利用q PCR技术对磁珠吸附的菌株进行检测。结果采用IMS-q PCR方法检测,当25 g样本中菌量为100cfu,样品被1∶2稀释时,检测结果呈阳性,检测全过程需时2~3 h。结论 IMS-q PCR检测O157∶H7大肠埃希菌的方法具有灵敏度高、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 method for rapid detection of enterohemorrhagic Escherichia coli O157: H7 by immunomagnetic separation (IMS) -prime real-time PCR (qPCR). Methods According to the specific gene rfb EO157 of enterohemorrhagic Escherichia coli O157: H7 antigen, primers and Taq Man probe were designed to establish q PCR system. The O157: H7 Escherichia coli in the sample was labeled with hemorrhagic large intestinal O157 antibody Immunomagnetic beads were used for specific capture, and q PCR was used to detect the beads adsorbed by the beads. The results of detection by IMS-q PCR method, when the 25 g sample in the amount of 100cfu, the sample was diluted 1: 2, the test results were positive, the whole test takes 2 ~ 3 h. Conclusion The method of IMS-q PCR detection of O157: H7 Escherichia coli has the advantages of high sensitivity, strong specificity and rapidity. It can be used in the rapid diagnosis of food poisoning caused by Escherichia coli O157: H7 and the detection of food microorganisms Molecular epidemiological investigation of the disease provides new means of detection.