肠侵袭性大肠杆菌DPO-PCR检测方法的建立与应用  

Development and application of DPO-PCR method for the detection of enteroinvasive Escherichia coli

作　　者：李丹丹[1] 徐义刚 邱索平 王昱[4] 高会江[5] 高慎阳[6] 

机构地区：[1]海南出入境检验检疫局检验检疫技术中心,海口570311 [2]黑龙江出入境检验检疫局检验检疫技术中心,哈尔滨150001 [3]从化出入境检验检疫局,从化510900 [4]重庆出入境检验检疫局检验检疫技术中心,重庆404100 [5]中国农业科学院北京畜牧兽医研究所牛遗传育种研究室,北京100193 [6]辽宁医学院畜牧兽医学院,锦州121001

出　　处：《中国人兽共患病学报》2015年第9期826-830,共5页Chinese Journal of Zoonoses

基　　金：海南省社会发展科技专项(2015SF29);国家质检总局科技项目(2013IK031;2013IK051;2015IK089);重庆市科技计划项目(cstc2014yykfA80017);海南省应用技术研究与开发专项项目(ZDXM20130025);广东检验检疫局科技计划项目(2011GDK44;2013GDK04)~~

摘　　要：目的为建立肠侵袭性大肠杆菌（EIEC）的DPO-PCR快速检测方法。方法本研究以EIECipaH基因为靶基因设计了一对双启动寡核苷酸（DPO）引物,建立了EIEC DPO-PCR检测方法。结果退火温度范围在47℃~67℃内都能有效地扩增出目的基因,表明该检测方法对退火温度不敏感;该DPO引物仅与EIEC的扩增反应呈阳性,而与其他菌株无非特异性扩增反应,表明该方法特异性强;该方法的灵敏度为1.17×102 cfu/mL。利用该检测方法对采集的230份样品进行检测,共计检出3份EIEC阳性样品,与国标法（GB 4789.6-2003）检测结果一致,显示了良好的实用性。结论该DPO-PCR方法设计简单、特异性强,具有良好的实用性。To establish a rapid assay for enteroinvasive Escherichia coli （EIEC） detection, a dual-priming oligonucleotide （DPO）-based PCR method was developed using the DPO primers targeting ipaH gene of EIEC. Results showed that annealing temperature range at 47-67 ℃ can effectively amplify the target gene, primers were designed to show that the annealing temper- ature was not sensitive; in addition, the DPO-PCR method had high specificity due to special structure of DPO primers, thus no non-specific amplifications were produced in reaction; sensitivity of the method was 1.17 × 102 cfu/mL. Furthermore, a total 3 positive samples for EIEC 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, simple, rapid and sensitive detection method for EIEC infection.

关 键 词：肠侵袭性大肠杆菌 IPAH基因 DPO-PCR 

分 类 号：S852.61[农业科学—基础兽医学]