检测牛感染犬新孢子虫的TaqMan-MGB Real-time PCR方法的建立
Journal of China Agricultural University(2016)
Abstract
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150bp,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。
MoreKey words
3D digital PCR,Real-time PCR,Neosporosis,Nc2 gene,TaqMan-MGB probes
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