牛新孢子虫二温式PCR检测方法的建立与应用  

Establishment and Application of Two-temperature Polymerase Chain Reaction for Detection of Neospora caninum in Cow

作　　者：季新成[1] 王文 牛国辉 郑培[1] 张玲[1] 

机构地区：[1]新疆出入境检验检疫局,新疆乌鲁木齐830063 [2]新疆动物防疫监督总站,新疆乌鲁木齐830060 [3]新疆计量测试研究院,新疆乌鲁木齐830011

出　　处：《动物医学进展》2011年第8期17-20,共4页Progress In Veterinary Medicine

基　　金：国家质量监督检验检疫总局科研项目(2009IK011)

摘　　要：根据已发表的犬新孢子虫种属特异性基因片段Nc-5基因序列设计合成一对特异性引物,采用均匀设计方法对引物浓度、Mg2+浓度、dNTP、Taq酶和退火温度进行了优化,建立了检测牛新孢子虫的二温式PCR方法。该方法对牛新孢子虫DNA的检测灵敏度为23.5 fg/反应。应用该方法对50份全血和8份流产胎儿样本进行了检测,有3份全血和1份流产胎儿样本为阳性。与三步法PCR相比较,该二温式PCR方法检测时间缩短了30 min,且方法具有快速、灵敏、准确、特异等优点,可用来对牛新孢子虫病的检测。A pair of specific primers were designed according to the published Nc-5 gene sequence of Neos-pora caninurn in GenBank. After the optimization of Mg^2＋ concentration, primers concentration, dNTP concentration, Taq enzyme and anneal temperate by uniform design method, a two-temperature PCR method was established to detect the infection of N. caninum. The sensitivity of the method to N. caninum was 23.5 fg reach reaction. 50 blood samples and 8 of fetal samples from abortion cows were detected by the method. Three blood samples and one fetal sample were conformed as positive. The detection time reduced 30 minutes compared with three step PCR, and the method was rapid, sensitive, accurate and specific for the detection of n. caninum .

关 键 词：新孢子虫 二温式PCR 均匀设计 牛 

分 类 号：S852.723[农业科学—基础兽医学] Q789[农业科学—兽医学]