摘要
为了快速检测牛源粪肠球菌,本实验建立了粪肠球菌esp基因实时荧光定量PCR标准曲线。首先参照esp基因在Genbank中的序列进行引物的设计与合成,利用PCR扩增目的基因并将其连接到pMD18-T载体上,并转化进入大肠埃希菌TOP10感受态细胞,经测序鉴定成功后,提取重组质粒并以10倍系列稀释后作为标准模板,进行实时荧光定量PCR。成功建立了牛源粪肠球菌esp基因荧光定量PCR标准曲线及其直线回归方程,所得熔解曲线峰值单一,表明引物具有很好的特异性;标准曲线相关系数r^2=0.9998,说明线性关系良好,目的基因的荧光定量PCR的标准曲线构建成功。本研究为快速检测牛源粪肠球菌奠定了基础。
In order to rapidly detect bovine enterococcus faecalis,this experiment constructed standard real-time fluorescent quantitative PCR curve of enterococcus faecalis esp.Firstly,we was synthesis of the primers by referring to the sequence of esp in Genbank.The target gene was amplified by PCR and connected to pMD18-T carrier,and then transformed into E.coli TOP10 competent cells.The recombinant plasmid which the successful sequencing identified was extracted and diluted by 10-fold series to be served as the standard template for real-time fluorescence quantitative PCR.Finally,the standard curve and linear regression equation of real-time fluorescence quantitative PCR of esp from bovine enterococcus faecalis were constructed.The single melting temperature peak corresponded to a specific product,revealing that the results were accurate and reliable.Moreover,the correlation coefficient(r2)of the standard curve was 0.9998,which showed that the linear relationship was good and the standard curve of the real-time fluorescence quantitative PCR of esp was successfully constructed.This study laid a foundation for the rapid detection of the bovine enterococcus faecalis.
出处
《中兽医学杂志》
2019年第11期13-15,共3页
Chinese Journal of Traditional Veterinary Science
基金
甘肃农业大学2019年学生科研训练计划(SRTP,201903046)
甘肃农业大学盛彤笙基金(GSAU-STS-1522)
甘肃省科技计划项目重点研发计划(18YF1NA077)
甘肃农业大学动物医学院教研产学支持项目(JYCX-KX004)。
关键词
粪肠球菌
ESP
实时荧光定量PCR
标准曲线
bovine enterococcus faecalis
esp
real-time fluorescence quantitative PCR
The standard curve
