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

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。

Abstract:

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、Mg²⁺ 、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;Mg²⁺concentration:3.75×10^-3mol/L;primer concentration 0.65×10^-6mol/L;probe concentration 0.30×10^-6mol/L;Cycle condition: step1:95℃,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.