新孢子虫核酸检测方法的建立及应用评价

以新孢子虫Nc-5基因为目的基因,建立检测新孢子虫的PCR方法并初步应用。根据Gen Bank发布的新孢子虫Nc-5特异性基因序列设计引物,以新孢子虫基因组DNA为模板,利用梯度PCR法优化反应条件,建立PCR检测方法。以弓形虫、疟原虫、牛环形泰勒虫、包拉米虫和马尔太虫为模板进行扩增以验证建立方法的特异性。采用紫外分光光度计测定新孢子虫基因组DNA浓度和纯度,倍比稀释后的DNA进行PCR扩增,产物电泳分析确定建立方法的敏感性;选择高浓度和低浓度的新孢子虫基因组DNA重复检测3次,分析建立方法的重复性和稳定性。利用该方法对实验室建立的小鼠感染模型进行初步应用,评价建立方法的检测效果。结果成功建立新孢子虫的核酸扩增检测方法,扩增序列与Gen Bank(LN714476.1)中Nc-5基因序列一致性为100%,建立的新孢子虫核酸扩增检测方法与弓形虫、疟原虫、牛环形泰勒虫、包拉米虫和马尔太虫均无交叉反应,最低能检测新孢子虫DNA浓度为0.5788 pg/μL,应用该方法检测感染新孢子虫的BALB/c小鼠模型,可在肺和脑组织中检测到新孢子虫的感染。结果表明,建立的新孢子虫核酸扩增检测方法有效,并为后续研究奠定基础。

ESTABLISHMENT OF NEOSPOROSIS NUCLEIC ACID DETECTION METHODS AND PRIMARY APPLICATION

Establish a highly specific and sensitive PCR method to detect Neospora caninum.A set of primers was designed by targeting the Nc-5 gene of Neospora caninum.The specificity was assayed by testing different species of parasites (i.g.Toxoplasma gondii,Plasmodium,Theileria annulata,Bonamia,Marteilia refringens).The sensitivity of the method was test by series of to dilute DNA of Neospora caninum.The detection effect of this method was evaluated by using the mouse infection model established in the laboratory.The assay indicates PCR method is a potential and valuable means for Neospora caninum detection.The detection sensitivity of the PCR method is about 0.5788 pg/μL DNA.Moreover this method has high specificity,repeatable and stability.