新孢子虫荧光PCR检测方法的研究

根据已知的犬新孢子虫种属特异性基因片段Nc-5基因序列,设计荧光定量PCR引物和荧光探针,经反应条件的优化,建立了检测新孢子虫的荧光定量PCR方法。该方法的检测灵敏度为10拷贝/反应。通过对系列稀释的重组质粒进行重复性检测,Ct值的变异系数为0.50%~1.18%。应用该方法对50份牛全血和8份流产胎儿样本进行检测,有5份全血和1份流产胎儿样本为阳性,阳性检出率均为10.3%,比普通PCR方法阳性检出率（7%）高。且具有较好的特异性和可重复性,可用来对新孢子虫病快速准确检测。

Research of Real-time PCR for Detection of Neospora Caninum

A pair of specific primers and probe were designed according to the published Nc-5 gene sequence of Neospora caninum （N.caninum） in GeneBank. After the optimization of Mg2＋ concentration, primer concentration, probe concentration, dNTP concentration, Taq enzyme and anneal temperature, a real-time PCR method was established to detect N.caninum infections. The sensitivity of the method for NC-5 recombinant plasmid was 10 copies per reaction. The coefficient of variation（CV）for repeated detection of serial dilutions of recombinant plasmid was 0.50%-1.18%.50 cattle blood sample and 8 cattle embryo samples of miscarriages were detected by the method, with 5 blood samples and 1 fetus sample as positive. The results showed that the average positive ratio was 10.3%, higher than the common PCR（7%）.The method had good specificity and reliability, it could be used to detect Neospora caninum.