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| 犬细小病毒荧光定量PCR检测方法的建立与评价 | |
| 引用本文： | 李英俊,史利军,李刚,侯绍华,曾妮,章金刚,朱鸿飞.犬细小病毒荧光定量PCR检测方法的建立与评价[J].中国兽医学报,2010,30(12). |
| 作者姓名： | 李英俊 史利军 李刚 侯绍华 曾妮 章金刚 朱鸿飞 |
| 作者单位： | 1. 军事医学科学院放射与辐射医学研究所,北京100850; 2. 中国农业科学院北京畜牧兽医研究所,北京100193; 3. 军事医学科学院野战输血研究所,北京100850; |
| 摘 要： | 通过序列比对和Blast分析,选定犬细小病毒(Canine parvovirus,CPV)VP2蛋白保守区基因为检测的目的基因,引物采用Primer Premier 5.0软件设计。利用灵敏度较高的TaqMan探针法建立CPV核酸检测方法。通过对标准品的扩增、测序及对标准扩增曲线的绘制,建立CPV核酸检测方法。同时对建立的检测方法进行了检测特异性、灵敏度和重复性分析。将阳性对照标准品进行10倍梯度稀释后可检测到102拷贝/μL样品,表明该检测体系具有较高的检测灵敏度。通过分析表明,本检测方法在用空白对照及类似的猪细小病毒、猪圆环病毒作为扩增对照时,没有发现非特异性产物的产生,表明该体系对于CPV的检测是特异的。通过6次批间重复检测,体系的变异系数小于3%,表明该检测体系具有良好的重复性。 |
| 关 键 词： | 犬细小病毒 VP2蛋白基因 实时荧光定量PCR |
Establishment and evaluation of real-time PCR for canine parvovirus detection |
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| LI Ying-jun,SHI Li-jun,LI Gang,HOU Shao-hua,ZENG Ni,ZHANG Jin-gang,ZHU Hong-fei.Establishment and evaluation of real-time PCR for canine parvovirus detection[J].Chinese Journal of Veterinary Science,2010,30(12). | |
| Authors: | LI Ying-jun SHI Li-jun LI Gang HOU Shao-hua ZENG Ni ZHANG Jin-gang ZHU Hong-fei |
| Abstract: | A rapid real-time polymerase chain reaction（RT-PCR） for detecting of canine parvovirus（CPV） was established.Primers were designed according to VP2 protein gene by Primer Premier 5.0.In response,a sensitive assay with the TanMan probe was developed and validated.Amplifying curve showed that this method could successfully amplify 102 copies/μL CPV gene.Meanwhile reference porcine parvoivrus（PPV）,porcine circovirus（PCV） and blank control were all negative.Ten-fold successive dilutions of positive CPV DNA were used to measure the sensitivity of RT-PCR.The assay system showed high reproducibility with CV3%.Thus the newly-built RT-PCR assay was indicated to be a rapid,sensitive and specific test for detecting CPV. |
| Keywords: | canine parvovirus VP2 gene real-time PCR |
| 本文献已被 CNKI 万方数据 等数据库收录！ | |