鹿布鲁氏菌实时荧光定量PCR检测方法的建立

Establishment of a real-time fluorescent quantitative PCR assay for Detection of Brucella of Deer

本研究依据布鲁氏菌的特异性基因Omp25c的部分片段作为靶基因,设计探针引物,且优化了反应体系,筛选出引物、探针的最优浓度配比。将扩增产物连接到PGEM-T载体上,制备标准品及标准曲线,建立鹿布鲁氏菌荧光定量PCR检测方法,并对其特异性、稳定性、敏感性进行评价。由标准曲线可知该方法的最低检测浓度可达到36拷贝/μL,比常规PCR灵敏度高出很多。

To aid in the development of a rapid assay to detect Brucella in deer,specific probe and primers was optimized based on the conservative Omp25c segment of Brucella and the reaction system was improved for selecting the best concentration of the primers and the probe. The amplified gene was connected to PGEM-T vector,and the developed RT-PCR assay was evaluated with regard to its specificity,replicability and sensitivity via constructing standard samples and standard curve. The result showed that the detecting limit was 36 copies/μL and more sensitive than the general PCR.