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| 检测牛感染犬新孢子虫的TaqMan-MGB Real-time PCR方法的建立 |
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陈千林1, 刘梦丽1, 许正茂1, 王振宝2, 吉尔格力3, 史亚明1, 巴音查汗1
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| (1.新疆农业大学 动物医学学院, 乌鲁木齐 830052;2.伊犁出入境检验检疫局综合技术服务中心, 新疆 伊宁 835000;3.新疆巴音郭楞职业技术学院 生物工程系, 新疆 库尔勒 841000) |
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| 摘要: |
| 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。 |
| 关键词: 新孢子虫病 Real-time PCR 3D数字PCR TaqMan-MGB探针 Nc2基因 |
| DOI：10.11841/j.issn.1007-4333.2016.12.08 |
| 投稿时间：2015-12-28 |
| 基金项目:新疆维吾尔自治区科技成果转化专项项目（201454129）；巴音郭楞蒙古自治州科学技术研究“星火”项目（201403）；库尔勒市重点科技计划项目（2013051108） |
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| Establishment of TaqMan-MGB Real-time PCR detection method of Neospora caninum in infected cattle |
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CHEN Qian-lin1, LIU Meng-li1, XU Zheng-mao1, WANG Zhen-bao2, GELI Ji-er3, SHI Ya-ming1, CHAHAN Ba-yin1
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| (1.College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China;2.Synthesis Technique Service Center of Yili Entry-Exit Inspection and Quarantine Bureau, Yining 835000, China;3.Department of Biological Engineering, Bayinguoleng Vocational and Technical College, Korla 841000, China) |
| Abstract: |
| 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. |
| Key words: Neosporosis Real-time PCR 3D digital PCR TaqMan-MGB probes Nc2 gene |
