摘要
为了建立一种能够快速检测毛皮动物源大肠埃希菌耶尔森菌强毒力岛(HPI)中irp2、FyuA毒力基因的二重PCR方法,根据GenBank中irp2、FyuA基因的序列,设计并合成两对引物,扩增得到irp2、FyuA基因片段,大小分别为300bp和951bp。据此建立双重PCR方法,并将反应条件进行了优化。结果表明,用所建立的二重PCR方法可同时特异性扩增出大肠埃希菌irp2、FyuA毒力基因片段,对沙门菌、金黄色葡萄球菌、铜绿假单胞菌、克雷伯菌均无特异性扩增。菌液最低检出量为2.8×105CFU/mL。在78份临床样品检测中,二重PCR检测结果与常规PCR检测结果一致。所建立的大肠埃希菌irp2、FyuA毒力基因二重PCR检测方法具有很好的特异性和灵敏度,能够提高临床样品检测的效率。
In order to establish a duplex PCR method for rapid detection of the virulence genes of irp2 and FyuA in the virulence island(HPI)of Escherichia coli in fur animals,the sequences were designed and synthesized according to the sequences of irp2 and FyuA genes in Genbank.Two pairs of primers were used to amplify the gene fragments of irp2 and FyuA,and the sizes were 300 bp and 951 bp,respectively.Based on this,a duplex PCR method was established and the reaction conditions were optimized.The results showed that the virulence gene fragments of E.coli irp2 and FyuA could be simultaneously and specifically amplified by the established duplex PCR method,and there was no specific amplification of Salmonella,Staphylococcus aureus,Pseudomonas aeruginosa and Klebsiella pneumoniae.The minimum detectable amount of broth was 2.8×10 5 cfu/mL.In the detection of 78 clinical samples,the results of the duplex PCR were consistent with the results of conventional PCR.The duplex PCR detection method for virulence genes of E.coli irp2 and FyuA established in this study has good specificity and sensitivity,and can improve the efficiency of clinical sample detection.
作者
高光平
朱利霞
王洪彬
赵希艳
王双月
GAO Guang-ping;ZHU Li-xia;WANG Hong-bin;ZHAO Xi-yan;WANG Shuang-yue(Hebei Key Laboratory of Preventive Veterinary Medicine,Hebei Normal University of Science & Technology,Qinhuangdao,Hebei,066600,China)
出处
《动物医学进展》
北大核心
2019年第6期12-16,共5页
Progress In Veterinary Medicine
基金
河北省现代农业产业体系肉牛产业创新团队疫病防控项目(HBCT2018130203)
河北省教育厅基金项目(SLRC2017037)
河北省高层次人才资助项目(A2016005046)
河北科技师范学院科学研究基金项目
