禽源性大肠杆菌HPI毒力岛irp2基因PCR检测方法的建立与应用

发布时间：2018-12-06 08:29

【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~(-4)ng/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。
[Abstract]:In order to understand the prevalence and gene characteristics of high virulence island (High pathogenicity island HPI) in clinical avian pathogenic Escherichia coli, a pair of specific primers were designed and synthesized according to the known irp2 gene sequence of Gen Bank, and a method for detection of irp2 gene PCR was established. To optimize and determine the characteristics of PCR amplification, to evaluate sensitivity, specificity and repeatability. Irp2 gene PCR and genetic variation analysis were performed in 256 clinical isolates of avian Escherichia coli. The results showed that the sensitivity of the established PCR assay was 2.14 脳 10 ~ (-4) ng/ 渭 L. There was no cross reaction with Salmonella, Haemophilus paraequis, Riemer Duck, Pasteurella, Mycoplasma, Newcastle disease virus and H9N5 nucleic acid. The reproducibility showed that 3 positive samples were repeated for 5 times, and the coefficient of variation was 3.45.00.256 clinical samples were tested for irp2 gene by PCR. The positive rate was 30.6%. The sequence of irp2 gene of 9 isolates was obtained, and the analysis showed that the homology of the known gene with GenBank was above 96.2%. The results showed that the established PCR method for detection of virulence island irp2 gene of avian Escherichia coli had good specificity, sensitivity and reproducibility, and could be used for rapid detection of virulence island gene in clinical pathogenic Escherichia coli.
【作者单位】：山东省滨州畜牧兽医研究院;山东省滨州畜禽蜂胶疫苗研究开发推广中心;山东绿都安特动物药业有限公司;
【基金】：山东省自然科学基金资助项目(ZR2014CQ012)
【分类号】：S852.612

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