布鲁氏菌实时定量荧光 PCR 检测体系的建立及应用

Establishment and application of real-time fl uorescence quantitative PCR system for the detection of Brucella spp

本根据GenBank上公布的布鲁氏菌BCSP31基因保守区域序列设计合成一对特异性引物与TapMan探针,建立整套快速准确鉴定布鲁氏菌的实时定量荧光PCR检测体系。以实验室构建的克隆有布鲁氏菌目的片段的重组质粒为标准品,进行实时定量荧光PCR反应检测,通过优化反应条件,建立标准曲线。以标准品为模板,对该方法的特异性、敏感性与重复性进行检测与分析。并以临床样本进行检测的结果进行比较分析,结果表明,该检测体系的检测灵敏度高,重复性与特异性良好。本研究建立的实时定量荧光PCR可用于准确检测样本中的少量的布鲁氏菌,具有良好的应用前景和市场价值。

According to the conserved sequences of Brucella BCSP31 genes, a pair of specific primers and probe were designed and synthesized to establish a rapid and accurate detection system for identification of Brucella by real-time quantitative PCR assay. The prepared Brucella recombinant plasmid containing target gene fragment was taken as the standard in the real-time quantitative fluorescent PCR, and the standard curve was then established. The specificity, sensitivity and reproducibility of this detection system were determined with standard as template. Comparison of clini-cal samples and standard samples showed that the PCR system was of high sensitivity, reproducibility and specificity. In conclusion, the study established real-time quantitative fluorescent PCR assay which could be used to detect small amount of Brucella in sample, with sound applicable prospect and quite good market value.