检测牛感染犬新孢子虫的TaqMan-MGB Real-time PCR方法的建立
Establishment of TaqMan-MGB Real-time PCR detection method of Neospora caninum in infected cattle

为建立牛感染犬新孢子虫的Real-time PCR检测方法，根据犬新孢子虫Nc2基因保守序列设计特异性检测引物和TaqMan-MGB探针，建立新孢子虫病TaqMan-MGB Real-time PCR检测方法。PCR扩增产物约为150 bp，与预期片段大小相符；Real-time PCR扩增表明，Ct值与梯度稀释的阳性质粒模板呈良好的线性关系；当检测牛源性弓形虫、牛环形泰勒和牛巴贝斯等虫种阳性DNA时均为阴性；经3D数字PCR判定，Real-time PCR方法的最低有效检测量为6.41拷贝/μL，灵敏性是常规PCR的1 000倍；重复性试验的组内平均变异系数为1.108%，组间为2.732%；本方法与《新孢子虫病检疫技术规范》（SN/T 3499-2013）的检测符合率为100%（46/46）。该Real-time PCR方法可用于早期诊断和日常监测家畜感染犬新孢子虫。
To establish Real-time PCR detection method of Neospora caninum in infected cattle,specific primers and TaqMan-MGB probes were designed according to N.caninum Nc2 conserved sequences.The size of PCR amplified fragment was about 150 bp and is consistent with the expected size;Real-time PCR amplification results showed that:The concentration of positive templates had a good linear relationship with the Ct value;The results were negative when detection positive DNA of Toxoplasma gondii,Theileria annulata,Babesia bovis and other parasites;Determination by 3D digital PCR,the lowest effective detection was 6.41 copy/μL,which was 1 000 times of the conventional PCR;The average coefficient variation between groups was 2.732% and was 1.108% within groups;And the detection result of this method if the was 100% (46/46) coincidence to the quarantine protocol for Neosporosis (SN/T 3499-2013).In conclusion,this method can be used for early diagnosis and routine monitor of livestock infected by Neospora caninum.