鹿布鲁菌分型实时荧光定量PCR检测方法的建立

Establishment of real-time fluorescent quantitative PCR method for detecting the types of Brucellain deer

鹿布鲁菌病主要是由牛型、羊型和猪型布鲁菌引起的,其他感染型少见,本研究依据布鲁菌的特异性基因BruAB20168、BMEII0466、BR0952上的部分高度保守片段设计猪型、牛型、羊型引物和探针,用于检测并区分鹿源布鲁菌的种型,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pUC57载体上,制备标准品及标准曲线,建立鹿布氏菌各分型荧光定量PCR检测方法,并对其特异性、稳定性、敏感性进行评价。由标准曲线敏感性可知该方法的最低检测浓度分别可达到12、9、8copies/μL,比常规PCR灵敏度高出很多。

Brucellosis in deer is induced by Brucella abortus ,Brucella ovis and Brucella suis, rarely by other Brucella. To develop rapid method for detection and classification of Brucellosis in deer, we designed probe primer of Brucella abortus,Brucella ovis and Brucella suis based on the specific BruAB20168,BMEII0466 and BR0952 segment and improved the reaction system for selecting the best percentage of concentration between primer and probe. The amplified aim gene was connected to pUC57 vector, meanwhile we evaluated specificity, replicability and sensitivity via constructing standard samples and curve. The research showed that the lowest detecting concentration was 12,9 and 8 copies/μL,respectively and more sensitive than general PCR.