禽致病性大肠杆菌毒力基因多重PCR方法的建立和应用

Multiplex PCR assay for detection of virulence genes in Avian pathogenic Escherichia coli

【目的】建立禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)黏附相关基因、侵袭及毒素相关基因、抗血清存活相关基因及铁转运相关基因的多重PCR方法,实现禽致病性大肠杆菌毒力基因的简便、快速检测。【方法】根据GenBank公布的基因序列,设计合成18对特异性引物,通过条件优化,建立四组多重PCR体系,并通过模板倍比稀释检测各组多重PCR的灵敏性。利用多重PCR检测100株APEC毒力基因的分布,验证多重PCR方法的可行性。【结果】根据PCR扩增片段大小判定,上述四组多重PCR体系均能同时扩增出该组中的各个毒力基因,且灵敏度分别为:103CFU、103CFU、105CFU、105CFU细菌和1ng、1ng、10ng、10ng DNA。100株APEC的毒力因子检测结果显示,多重PCR和单基因PCR结果一致。【结论】建立的四组多重PCR方法能够简便、快速地检测禽致病性大肠杆菌的毒力基因,可用于毒力基因的鉴定以及流行病学调查。

[ Objective ] To efficiently study the virulence genes distribution of Avian pathogenic Escherichia coli （APEC）, we developed four multiplex PCR to detect adbesin-associated genes, invasin and toxin-associated genes, serum resistance- associated genes and iron acquisition-associated genes. [ Methods] According to gene sequences published in GenBank, we designed and synthesized 18 specific primer pairs, which were used in the four multiplex PCR. Then, we determined the sensitivity of multiplex PCR using diluted bacterial or DNA templates. To verify the feasibility of these multiplex PCR, we determined the distribution of virulence genes in 100 APEC isolates using these multiplex PCR. [ Results] According to the results of PCR, we can conclude that each of the 18 genes was exactly and effectively amplified in the four multiplex PCR. The sensitivities of these four multiplex PCR were 103 Colony forming units （CFU）, 103CFU, 105CFU, 105CFU bacteria and lng, lng, 10ng and 10ng DNA, respectively. Furthermore, the results multiplex PCR for virulence genes distribution in 100 APEC were same as the single PCR. [ Conclusion] These results suggest that multiplex PCR developed in this study could efficiently detect the virulence genes of APEC, which was a useful and rapid technique for epidemiological investigation.