实时荧光定量PCR检测沙门菌flor基因和sul2基因方法的建立  

Establish the detecting mothed of Salmonella flor and sul2 genes by Real-Time quantitative PCR

作　　者：曹堃[1] 王元兰[1] 刘宗梁[1] 刘军军[1] 魏建忠[1] 孙裴[1] 李郁[1] 

机构地区：[1]安徽农业大学动物科技学院安徽省畜禽产业共性研究院,合肥230036

出　　处：《中国抗生素杂志》2015年第7期531-537,共7页Chinese Journal of Antibiotics

基　　金：安徽农业大学学科学位点建设项目(No.XKXWD2013006);安徽农业大学大学生创新基金项目(No.2011053)

摘　　要：目的建立一种fl or基因和sul2基因的SYBR Green I实时荧光定量PCR方法,为检测沙门菌的耐药性以及耐药基因分子流行病学调查奠定基础。方法以氟苯尼考、复方磺胺甲噁唑抗性的沙门菌菌株为材料,根据Gen Bank中登陆的沙门菌fl or和sul2基因序列,设计并合成引物,建立基于SYBR Green I染料技术的Real-Time PCR检测体系,绘制出标准曲线并对其溶解曲线进行分析。结果以pMD18-T为载体构建的标准品的Ct值与样品的浓度的对数在一定范围内呈现良好的线性关系,两基因的检测域值较小,fl or基因可以检测到102 copies/μL的样品,sul2基因可以检测到103 copies/μL的样品,经耐药性与基因表达量相关分析,耐药性较高的菌株其fl or基因、sul2基因表达量也较高。结论建立的检测沙门菌中fl or基因和sul2基因的荧光定量PCR方法具有很好的灵敏性、特异性和重复性,可以广泛用于临床中fl or和sul2两种基因的检测以及沙门菌耐药分子流行病学的调查。Objective To establish a SYBY Green I real-time quantity PCR mothed to detect theflor and sul2 genes and lay foundations for detect the resistance of Salmonella and molecular epidemiological survey. Methods Salmonella resistance to florfenicol and trimethoprim and sulphame-thoxazole was material. According to the Salmonellaflor gene and sul2 gene sequence in GenBank, the specific primers were designed and synthesized, then established the Real-Time PCR SYBR Green I detection system on the techology of SYBR Green I dye. Results The Ct value of standard constructed by pMD 18-T vector has a good liner relationship with the logarithm of concentrains of the standard in a certain range. The dection threshod of two genes were small, forflor gene, 10^2copies/μL can be detected, for sul2 gene, 10^3copies/μL can be detected, the drug resistance and gene expression analysis shows the higher sistance strains offlor and sul2, the gene expression was higher. Conclusion The methods were established for detectflor and sul2 genes has good sensitivity, spectificity and reproducibility, it can be used in clinical detection offlor and sul2 genes and investigation of Salmonella resistant molecular epidemiology.

关 键 词：沙门菌 实时荧光定量PCR flor基因 sul2基因 

分 类 号：R378[医药卫生—病原生物学]