实时荧光定量-聚合酶链反应检测食品中金黄色葡萄球菌方法的研究

徐德顺 韩健康 吴晓芳

徐德顺, 韩健康, 吴晓芳.  实时荧光定量-聚合酶链反应检测食品中金黄色葡萄球菌方法的研究[J]. 疾病监测, 2009, 24(7): 541-544. doi: 10.3784/j.issn.1003-9961.2009.07.023
引用本文: 徐德顺, 韩健康, 吴晓芳.  实时荧光定量-聚合酶链反应检测食品中金黄色葡萄球菌方法的研究[J]. 疾病监测, 2009, 24(7): 541-544. doi: 10.3784/j.issn.1003-9961.2009.07.023
XU De-shun, HAN Jian-kang, WU Xiao-fang. Study of TaqMan assay of real-time fluorescence quantitative PCR to detect Staphylococcus aureus in food[J]. Disease Surveillance, 2009, 24(7): 541-544. doi: 10.3784/j.issn.1003-9961.2009.07.023
Citation: XU De-shun, HAN Jian-kang, WU Xiao-fang. Study of TaqMan assay of real-time fluorescence quantitative PCR to detect Staphylococcus aureus in food[J]. Disease Surveillance, 2009, 24(7): 541-544. doi: 10.3784/j.issn.1003-9961.2009.07.023

 实时荧光定量-聚合酶链反应检测食品中金黄色葡萄球菌方法的研究

doi: 10.3784/j.issn.1003-9961.2009.07.023

Study of TaqMan assay of real-time fluorescence quantitative PCR to detect Staphylococcus aureus in food

  • 摘要: 目的利用实时荧光定量-聚合酶链反应(real-time PCR)技术,建立食品中金黄色葡萄球菌(金葡萄)污染的快速敏感特异的检测方法。方法以金葡菌的IFemB/I基因作为靶序列,设计一对引物和探针,以金葡菌菌株提取核酸DNA作为模板,优化引物和探针的浓度比和Mgsup2+/sup浓度,以金葡菌和10种相关细菌考核检测体系的灵敏性、稳定性和特异性。并初步应用于样品的检测。结果本研究建立的反应体系在引物和探针的浓度为0.8 mol/L、0.6 mol/L,Mgsup2+/sup浓度为3.5 mmol/L时,具有良好的特异性和敏感性。在10株相关菌株的检测中,除金葡菌出现很好的阳性外,其余菌株均为阴性。在纯菌条件下,最低检测限为44 cfu/ml。稳定性分析表明:同一样品重复检测3次IC/It值的变异系数均5%。检测样品结果显示real-time PCR方法较传统方法敏感、快捷、简便。结论该方法特异性强,稳定性高,操作简便快捷,适应食品微生物检验发展需要,具有较大的推广及应用价值。
  • [1]

    [1]Atanassova V,Ring MA.Prevalence of <I>Staphylococcus aureus</I> and staphylococcal enterotoxins in raw pork and uncooked smoked ham-a comparison of classical culturing detection RFLP-PCR[J].<I>Int J Food Microbiol,</I> 2001,68:105-113.
    [2]Dinges M, Orwin PM,Patrick M. Schlievert Exotoxins of Staphyococcus aureus[J]. <I>Clin Microbiol Rev,</I> 2000,13(1):16-34.
    [3]Chen B, Huang XR, Tang MY, et al. Study on the rapid detection of <I>Staphylococcus aureus</I> with gene probe method in foodstuffs [J]. <I>Modern Preventive Medicine,</I> 2007,34 (6):1017-1021. (in Chinese)
    陈彬,黄晓蓉,汤敏英,等.基因探针法快速检测食品中金葡菌的研究[J]. 现代预防医学,2007,34 (6):1017-1021.
    [4]National standard of the Peoples Republic of China  Microbiological examination of food hygiene detection GB/T 4789.10-2003.Beijing: <I>Standards Press of China,</I> 2004. (in Chinese)
    中华人民共和国国家标准.食品卫生微生物学检验GB/T 4789.10-2003.北京:中国标准出版社,2004.
    [5]Mackay IM,  Andren KE,  Nische A. real-time PCR in virology[J].<I>Nucleic Acids Res,</I> 2002,30:1292-1305.
    [6]Cremonesi P,Luzzana M,Brasca M, et al. Development of a multiplex PCR assay for the identification of <I>Staphylococcus aureus</I> enterotoxigenic strains isolated from milk and dairy products[J]. <I>Mol Cell Probes,</I> 2005,19(5):299-305.
    [7]Klotz M,Opper S,Heeg K,et a1.Detection of <I>Staphylococcus aureus</I>  enterotoxins A to D by real-time fluorescence PCR assay[J].<I>J Clin Microbiol,</I>2003,10(41):4683-4687.
    [8]Florkowski CM.Sensitivity, Specificity, Receiver Operating Characteristic (ROC) Curves and Likelihood Ratios: Communicating the Performance of Diagnostic Tests[J]. <I>Clin Biochem Rev,</I>2008,29(Suppl 1):S83-87.

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出版历程
  • 收稿日期:  2008-08-01
  • 刊出日期:  2009-07-30

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