鉴定小肠结肠炎耶尔森氏菌及其毒力基因的多重PCR检测方法的建立

Detection of Yersinia enterocolitica and its virulence genes by a novel multiplex PCR

针对小肠结肠炎耶尔森氏菌的高度保守基因foxA以及3种毒力基因ail、ystA、ystB设计并合成引物,建立一种多重PCR方法,该方法能够鉴定小肠结肠炎耶尔森氏菌,同时检测其位于染色体上的毒力基因。用单重PCR方法和所建立的多重PCR方法对145份牛粪可疑样品进行检测并加以分析。结果显示,建立的多重PCR方法重复性良好,特异性强,灵敏度高,对ystA、ail、ystB以及foxA基因的最低检测限依次为3×10^-3ng/μL、3×10^-3ng/μL、1.8×10^-3ng/μL和3.2×10^-3 ng/μL。对145份牛粪可疑样品的检测结果显示,其中38份样品只含有foxA基因,71份样品含有foxA和ystB基因,未检出含有ail和ystA基因的样品,与单重PCR结果一致。该方法可一次性检测4种基因,较单重PCR方法更省时省力,对于含有不同毒力基因的小肠结肠炎耶尔森氏菌的快速检测具有重大意义。

In order to detect Yersinia enterocolitica and its chromosome virulence genes,a multiplex PCR assay was established.The ail,yst A,yst B genes and the highly conserved gene fox A of Yersinia enterocolitica were used to design the primers of this method.145 suspicious cattle dung samples were detected and analyzed by single-plex PCR and established multiplex PCR.The Multiplex PCR assay showed good specificity and the detection minimum of yst A,ail,yst B and fox A gene by the PCR was 3×10^-3 ng/μL,3×10^-3 ng/μL,1.8×10^-3 ng/μL and 3.2×10^-3 ng/μL,respectively.The results of 145 suspicious samples of cattle dung showed that 38 samples contained only fox A gene,71 samples contained fox A and yst B genes,and no samples contained ail and yst A genes,which was consistent with the results obtained by Single PCR.This assay has great significance for the rapid detection of Yersinia enterocolitica with different virulence genotypes.