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.
出处
《微生物学通报》
CAS
CSCD
北大核心
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)
