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致病性大肠埃希菌和沙门菌毒力岛基因的双重PCR检测方法的建立
引用本文:邹兰,祁克宗,薛秀恒,汪雪雁. 致病性大肠埃希菌和沙门菌毒力岛基因的双重PCR检测方法的建立[J]. 中国微生态学杂志, 2012, 24(6): 504-507
作者姓名:邹兰  祁克宗  薛秀恒  汪雪雁
作者单位:安徽农业大学茶与食品科技学院,安徽合肥,230036
基金项目:国家自然科学基金(30871851)
摘    要:目的实现对致病性大肠埃希菌(E.coli)、沙门菌(Salmonella)的同时检测,建立快速灵敏的双重PCR检测方法。方法以致病性大肠埃希菌和沙门菌毒力岛基因为研究对象,根据GenBank发表的大肠埃希菌和沙门菌毒力岛基因序列,分别设计合成了大肠埃希菌毒力岛irpl、irl)2和fyuA,沙门菌毒力岛mgtC、sseL和sopB等6对引物,以禽致病性大肠埃希菌(CVCC1565)菌株和沙门菌(ATCC9150)菌株的核酸混合物为模板,经引物特异性试验,引物组合,成功建立了快速鉴别检测致病性大肠埃希菌和沙门菌的双重PCR方法。结果特异性试验结果显示,引物irpl、irp2和fyuA仅能扩增出大肠埃希菌(CVCC1565)的特异性片段,大小分别是799、414和948bp;引物mgtC、sseL和sopB仅能扩增出沙门菌(ATCC9150)的特异性片段,大小分别是500、269和1000bp。敏感性试验结果表明大肠埃希菌和沙门菌的最低检测限分别为2.2×101CFU/mL和2.0×101CFU/mL。结论本研究建立的双重PCR方法具有特异性强、敏感性高、快速简便等特点,可用于致病性大肠埃希菌和沙门菌的联合检测与鉴别诊断。

关 键 词:大肠埃希菌  沙门菌  毒力岛  双重PCR

Establishment of multiplex PCR assay for the detection of pathogenicity island on E.coli and Salmonella
ZOU Lan , QI Ke-zong , XUE Xiu-heng , WANG Xue-yan. Establishment of multiplex PCR assay for the detection of pathogenicity island on E.coli and Salmonella[J]. Chinese Journal of Microecology, 2012, 24(6): 504-507
Authors:ZOU Lan    QI Ke-zong    XUE Xiu-heng    WANG Xue-yan
Affiliation:(College of Tea & Food Science and Tcehnology,Anhui Agrecultural University,Hefei 230036,China)
Abstract:Objective To set up a sensitive,specific multiplex PCR assay so as to achieve quick detection of the pathogenic E.coli and Salmonella at the same time.Method From the pathogenicity island of E.coli and Salmonella,six pairs of primers irp1,irp2,fyuA and mgtC,sopB,sseL were designed according to the genomic sequences of E.coli and Salmonella available in GenBank Genomic DNA of E.coli(CVCC 1565) and Salmonella(ATCC 9150) strains.They were then mixed for amplification to establish a multiplex-PCR for detection of the pathogenicity island on E.coli and Salmonella through primer specific test and primer-assembly.Result Using the primers irp1,irp2,and fyuA,only the specific 799 bp,414 bp,and 948 bp fragments were amplified from DNA samples of E.coli(CVCC 1565);Using the primers mgtC,sopB,and sseL,only the specific 500 bp,269 bp,and 1000 bp fragments were amplified from DNA samples of Salmonella(ATCC 9150).The sensitivity test showed that DNA used for the multiplex-PCR might be as low as 2.2×101 CFU/mL for E.coli(CVCC 1565) and 2.0×101 CFU/mL for Salmonella(ATCC 9150),respectively.Conclusion This multiplex-PCR assay is rapid,sensitive and specific for simultaneous diagnosis or differentiation of E.coli and Salmonella.
Keywords:E.coli  Salmonella  Pathogenicity island  Multiplex-PCR
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