| 牛新孢子虫内标双重荧光PCR检测方法的建立 |
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| 引用本文： | 季新成,段晓东,黄玲,牛国辉,郑培,刘小兰.牛新孢子虫内标双重荧光PCR检测方法的建立[J].中国兽医学报,2012,32(3):406-410. |
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| 作者姓名： | 季新成 段晓东 黄玲 牛国辉 郑培 刘小兰 |
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| 作者单位： | 1. 新疆出入境检验检疫局,新疆乌鲁木齐,830063
2. 新疆计量测试研究院,新疆乌鲁木齐,830011 |
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| 基金项目： | 国家质量监督检验检疫总局科研基金资助项目(2009IK011) |
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| 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。
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| 关 键 词： | 犬新孢子虫 内标 双重荧光PCR |
Establishment of diplex real-time PCR system with internal control for detection of Neospora caninum |
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| JI Xin-cheng,DUAN Xiao-dong,HUANG Ling,NIU Guo-hui,ZHENG Pei,LIU Xiao-lan.Establishment of diplex real-time PCR system with internal control for detection of Neospora caninum[J].Chinese Journal of Veterinary Science,2012,32(3):406-410. |
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| Authors: | JI Xin-cheng DUAN Xiao-dong HUANG Ling NIU Guo-hui ZHENG Pei LIU Xiao-lan |
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| Institution: | 1(1.Xinjiang Exit-Import Inspection and Qurantin Bureau,Urumqi 830063,China;2.Xinjiang Uygur Autonomous Region Research Institute of Measurement & Testing,Urumqi 830011,China) |
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| Abstract: | 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. |
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| Keywords: | Neospora caninum internal control diplex real-time PCR |
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