禽源大肠杆菌耶尔森氏菌强毒力岛核心基因的检测及其irp2和int基因同源性

Core genes of high-pathogenicity island in avian Escherichia coli isolates

【目的】为了提高禽源大肠杆菌中耶尔森氏菌强毒力岛(HPI)的检测效率,了解高分子量铁调节蛋白2基因(irp2)和整合酶基因(int)在不同株禽源HPI+大肠杆菌间的同源性,进一步揭示禽源大肠杆菌HPI的转移规律。【方法】利用L16(44)正交试验设计,建立针对HPI核心基因irp2和fyuA的双重PCR,运用双重PCR方法检测禽源大肠杆菌临床分离株,并对检出的7株HPI阳性(HPI+)大肠杆菌进行irp2和int基因测序及同源性分析,同时结合这7株大肠杆菌的ERIC-PCR分析结果,对比分析int基因的分布特点。【结果】结果显示,新建立的双重PCR能特异性扩增出HPI核心基因;ERIC-PCR分析显示,HPI+大肠杆菌间差异均大于5%;HPI+大肠杆菌irp2基因高度保守(同源性大于99%),而int基因虽然都位于asn-tRNA位点,但基因序列在部分菌株间存在较大差异。【结论】建立了一种可以用于HPI的流行病学调查和实验室诊断的双重PCR方法,并推测区域外同源重组可能是HPI基因在大肠杆菌间水平转移的主要方式。

[Objective] In order to improve the detection efficiency of high-pathogenicity island （HPI） in avian Escherichia coli isolates, investigate the homology of its int genes and irp2 genes, and finally reveal the transfer regularity of HPI in avian E. coli. [Methods] Orthogonal experimental design was used to optimize multiple-PCR amplification system for irp2 and fyuA, which belong to the core genes of HPI, and avian E. coli clinical isolates were detected with this method. The genetic homologies of irp2 and int genes amplified from seven detected HPI-positive E. coli strains were analyzed. The distribution characteristic of int was analyzed comparing with the ERIC-PCR result of the seven HPI-positive E. coli strains. [Results] The core genes of HPI were specifically amplified by the multiple-PCR method. ERIC-PCR analysis shows that the degree of difference between the HPI-positive strains was higher than 5%. A great conservation （higher than 99% homology） of irp2 genes in HPI-positive E. coli strains was found, and all of the int genes were located in the vicinity of asn-tRNA, but not all of them were highly conserved. [Conclusion] We have established a multiple-PCR method which can be applied to the laboratory diagnosis test and epidemiological investigation of HPI, and suppose that the way of HPI horizontal transfer may be mainly based on the homologous recombination in the HPI-adjacent sequences.