【摘要】 为了解临床禽源致病性大肠杆菌中高致病性毒力岛（High pathogenicity island HPI）流行情况和基因特征,根据Gen Bank已知禽源irp2基因序列,设计、合成一对特异性引物,建立irp2基因PCR检测方法,优化、确定PCR扩增特性,进行敏感性、特异性、重复性检测评价。对256份临床分离禽源大肠杆菌进行irp2基因PCR检测和基因遗传变异分析。结果显示,建立的PCR检测方法敏感性可达2.14×10^-4ng/μL;特异性显示与鸡沙门菌、副鸡嗜血杆菌、鸭疫里默菌、禽巴氏杆菌、鸡毒支原体、鸡新城疫病毒、禽流感病毒（H9N5）核酸均无交叉反应;重复性显示3份阳性样品重复5次检测,变异系数3.4%⁵.0%。256份临床样品PCR检测irp2基因,阳性率30.6%。获得了9株分离株irp2基因序列,分析显示,与GenBank已知基因同源性高达96.2%以上。说明建立的禽源大肠杆菌HPI毒力岛irp2基因PCR检测方法具有良好的特异性、敏感性和重复性,可应用于临床致病性大肠杆菌毒力岛基因快速检测。更多还原

【Abstract】 The purpose of this paper is to establish the PCR detection method of irp2. To understand the clinical poultry pathogenic escherichia coli High pathogenicity island( HPI) prevalence and genetic traits,a pair of specific primers were designed and synthesised according to gene sequence of irp2 and the PCR detection method was established,evaluation about optimization,determining the PCR amplification characteristics,sensitivity,specificityor and repeatability were studied. Irp2 gene was detected and genetic variation was analyed on 256 E. coli strains isolated from clinical avian. Results showed that the sensitivity was 2. 14 × 10-4ng/μL; Specificity displayed that there was no cross reaction between salmonella,vice chicken haemophilus,Riemerella anatipestifer,poultry pasteurella,Mycoplasma gallisepticum,Newcastle disease virus,and avian influenza virus( H9N5); Repetitive showed that the variation coefficient was 3. 4 ~ 5. 0% if three positive samples repeated 5 times testing. Irp2 gene was detected by PCR from 256 clinical samples,and positive rate was 30. 6%. The analysis of 9 strain irp2 gene sequences revealed that the homology was more than 96. 2% when compared with Genbank known gene. The method of E. coli HPI irp2 gene PCR detection has a good specificity,sensitivity and repeatability,and it can be applied to clinical gene rapid detection of HPI.更多还原