摘要
为建立牛新孢子虫的快速准确检测方法,根据犬新孢子虫种属特异性基因Nc-5序列,设计高度保守的引物和荧光探针,通过引物设计和搭桥PCR法扩增,获得Nc-5荧光PCR内标模板。对内标模板的添加量和反应条件进行优化,建立了牛新孢子虫内标双重荧光PCR检测体系。该方法具有较好的特异性;可以检测到10个拷贝/PCR反应的核酸分子,与不加内标的荧光PCR检测灵敏度相当;通过对系列稀释的核酸样品的重复性检测,变异系数为0.50%～1.30%。通过对58份临床样品分别用该方法、不含内标的荧光PCR方法和普通PCR方法检测,结果显示,该方法与不含内标的荧光PCR方法的阳性检出率均为10.3%,比普通PCR方法阳性检出率(7.0%)高;表明该方法可用于临床样品中牛新孢子虫的快速检测,并能对实验室进行质量控制。
To rapidly and exactly detect Neospora caninum（N.caninum） in bovine,high conservative primers and fluorescent probe were designed according to the published N.caninum Nc-5 gene sequence.The internal control（IC） templet of real-time PCR was achieved by amplifying with bridge-building PCR method.By optimizing the quantity of the IC templet and the reaction condition,the real-time PCR detection system with IC was established.The method was specific,and the detection limit was about 10 copies/PCR reaction,it was almostly coincident with the conventional real-time PCR without IC.The coefficient of variation（CV）to the detection of series dilution Nc-5 gene was 0.50%-1.30%,demonstrated the method was high reproducibility.58 clinical samples were detected with the real-time PCR with IC and without IC,the results showed that the average positive ratio both were 10.3%,higher than common PCR（7.0%）.Moreover,the statistics results indicated that there were PCR inhibition in blood specimens.It suggested that this method was useful in the rapid detection of Neospora caninum and lab quality control.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2012年第3期406-410,共5页
Chinese Journal of Veterinary Science
基金
国家质量监督检验检疫总局科研基金资助项目(2009IK011)
关键词
犬新孢子虫
内标
双重荧光PCR
Neospora caninum
internal control
diplex real-time PCR
