肠致病性大肠杆菌DPO-PCR快速检测方法的建立

Development of a dual-priming oligonucleotide-based PCR assay for the rapid detection of enteropathogenic Escherichia coli

为建立肠致病性大肠杆菌（EPEC）的快速检测方法,本研究以EPEC bfp A基因为靶基因设计了一对双启动寡核苷酸引物（DPO）,建立了EPEC的DPO-PCR快速检测方法。结果显示,退火温度在45℃~65℃范围内均能够高效地扩增出目的基因,表明DPO引物对退火温度不敏感。该方法对其他菌株的扩增结果均为阴性,特异性强;其灵敏度为97 cfu/m L。利用该方法和国标法分别对采集的230份临床样品进行检测,均共计检出5份EPEC阳性样品,两者符合率为100%。本研究建立的DPO-PCR方法设计简单、特异性强、灵敏度高,具有良好的实用性,为快速准确检测EPEC提供新的检测手段。

To establish a rapid assay for enteropathogenic Escherichia coli（EPEC） detection, a dual-priming oligonucleotide（DPO）-based PCR method was developed using the DPO primers targeting bfp A gene of EPEC. The DPO-PCR method had high specificity to detect EPEC due to special structure of DPO primers, thus no non-specific amplifications were produced from other bacteria, which was able to efficiently amplify target gene in the annealing temperature range from 45 ℃ to 65 ℃, showing insensitivity to annealing temperature. In addition, The DPO-PCR method had a detection limit of 97 cfu/m L for EPEC,Furthermore, five positive samples for EPEC were detected from 230 clinical samples by the DPO-PCR method, which was in accordance with the testing result by GB/T 4789.6-2003 standard detection protocol. Therefore, the DPO-PCR method provides a novel rapid and sensitive detection method for EPEC infection.