沙门氏菌荧光实时定量PCR检测试剂的研制及应用  

Research and Application on Detection of Salmonella sp. by FQ-PCR

作　　者：李光伟[1] 邱杨[1] 肖性龙 詹少彤[1] 兰敏[1] 黄伟[1] 周润华[1] 姚小文[1] 冯小军[1] 林涛[1] 梅艳群[1] 

机构地区：[1]东莞出入境检验检疫局,东莞523071 [2]深圳太太基因工程有限公司,深圳518057

出　　处：《微生物学通报》2007年第3期496-499,共4页Microbiology China

基　　金：东莞市科技局科研发展专项基金项目(No.2005D043)

摘　　要：荧光实时定量PCR技术是近年来广泛应用于沙门氏菌快速检测的现代方法之一,本研究建立了检测沙门氏菌快速、敏感、特异以及准确定量的FQ-PCR方法。采用沙门氏菌fimY基因序列,设计特异引物和探针,通过对Taq酶、Mg2+和引物探针浓度等反应体系和条件的优化,然后进行特异性和适用性实验。最优化结果为:Taq酶用量2.5U;Mg2+浓度为3.75×10-3mol/L;引物浓度为0.65×10-6mol/L,探针浓度为0.30×10-6mol/L;循环条件为step1:95℃2min,step2:95℃5s,60℃40s,40cycles。结果表明该FQ-PCR检测试剂具有快速、简单、灵敏度高、特异性强和适用范围广等优点,可应用于食品卫生监管、商品检验检疫以及临床诊断等领域。The FQ-PCR has been introduced for the Salmonella spp. detection these years. To develop a FQ-PCR assay to rapid,sensitive, specific and accurate for quantitative detect Salmonella spp. A pair of specific primers and probe were designed from the fimY gene of Salmonella spp. in this research. Through optimizing the Taq enzyme, Mg^2＋ , primer concentration and probe concentration system and condition of FQ-PCR, the detection sensitivity was improved dramatically. The results of optimum experiment as following： Taq enzyme ： 2.5U ; Mg^2＋ concentration： 3.75 × 10^-3 tool/L; primer concentration 0.65 × 10^-6 tool/L; probe concentration 0. 30 × 10^-6 tool/L; Cycle condition： stepl ： 95℃, 2min, step2： 95℃ 5s, 60℃ 40s,40cycles. Not only is this detection technology more specific, sensitive and efficient, but also the processing is wide application, rapid and simple. This FQ-PCR detection technology, therefore, will be used to detect Salmonella spp. for those areas food sanitation supervision, commodity inspection and detection,clinical diagnosis, etc.

关 键 词：沙门氏菌 荧光实时定量-PCR 检测 研制 

分 类 号：TS207.4[轻工技术与工程—食品科学]