肠出血性大肠杆菌O157∶H7 EMA-PCR检测技术的建立

Method to Detect Enterohemorrhagic Escherichia Coli O157∶H7 by EMA-PCR

肠出血性大肠杆菌O157∶H7是一种重要的人畜共患传染病病原菌。为建立一种特异、灵敏的O157∶H7新型检测技术,以O157抗原基因(rfbE基因)为模板设计特异性引物,利用叠氮溴化乙锭(ethidium monoazide bromide,EMA)处理菌液,沸水浴法制备细菌裂解液,优化PCR条件,建立一种快速、有效的O157∶H7活菌EMA-PCR检测方法。结果显示:EMA-PCR可从rfbE基因阳性菌株CVCC248和两株临床分离菌株cd0912、cd0803中扩增出大小为495 bp的特异性条带,检测灵敏度可达12 CFU/mL。经EMA处理,从含有1%～100%O157∶H7 CVCC248活菌混合悬液制备的DNA中均可扩增出目的片段。因此,成功建立了肠出血性大肠杆菌O157∶H7的EMA-PCR检测方法;该方法可避免因分析的样品中含有死细菌而造成的假阳性检测结果,检测的准确性和真实性较传统PCR大大提高。O157∶H7 EMA-PCR技术的建立为O157∶H7的临床诊断提供了新的方法,具有重要的实际应用价值和良好的应用前景。

Enterohemorrhagic Escherichia coli O157：H7 is one important zoonosis infectious disease pathogens. In order to establish a specific, sensitive new detection technology for O157 ：H7,the specific primers were designed according to the O157 antigen gene （rfl×E gene） as a template, the bacteria was treated with EMA, the bacterial lysates was prepared through the boil- ing water bath method, and PCR conditions were optimized, and a rapid and effective detection method of EMA-PCR for live O157：H7 was established. The results showed that the 495 bp amplicon was specifically detected using the EMA-PCR in posi- tive strains CVCC248 and two clinical isolates cd0912 and cd0803. The detection sensitivity was up to 12 CFU/mL. By EMA treatment, the target fragment was amplified from the DNA of mixed suspension containing 1%-100% live O 157：H7 CVCC2g8. Therefore,the EMA-PCR method for O157：H7 was successfully established. This method avoided the false positive test result caused by the sample containing dead bacteria, and improved the accuracy and authenticity of the detection in comparing with the conventional PCR. The EMA-PCR method for O157 ：H7 provides a new approach in the clinical diagnosis, it has important practical value and good prospect.