毛皮动物源大肠埃希菌毒力基因二重PCR检测方法的建立
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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300 bp和951 bp。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2.8×10~5 CFU/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.
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.
引文
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[3] 王艳萍,董林,郭时金,等.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[J].中国兽医杂志,2017,53(1):86-89.
[4] 闻晓波,崔玉东,朱战波,等.牛产肠毒素大肠杆菌毒力因子多重PCR检测方法的建立[J].中国兽医学报,2007,27(3):322-324.
[5] 徐香.肠毒素性大肠杆菌菌毛的快速检测方法[J].中国人兽共患病学报,1987,3(3):40-42.
[6] Koczura R,Kaznowski A.Occurrence of the Yersinia high-pathogenicity island and iron uptake systems in clinical isolates of Klebsiella pneumoniae[J].Microb Pathog,2003,35(5):197-202.
[7] Carniel E.The Yersinia high-pathogenicity island:an iron-uptake island[J].Microb Infection,2001,3(7):561-569.
[8] 刁有祥,李久芹,陈庆普.山东省鸡大肠杆菌的分离鉴定[J].中国预防兽医学报,2002,24(1):21-23.
[9] 丁天翊,潘阳阳,何翃闳,等.快速检测奶牛乳房炎四种病原菌的多重PCR方法的建立及应用[J].中国兽医科学,2018,48(1):19-26.