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| TaqMan荧光定量PCR检测猪流行性腹泻病毒方法的建立与初步应用 | |
| 引用本文： | 刘邓,袁秀芳,冉多良,徐丽华,牛登元,王朝文,王一成.TaqMan荧光定量PCR检测猪流行性腹泻病毒方法的建立与初步应用[J].中国兽医寄生虫病,2010,18(1):28-34. |
| 作者姓名： | 刘邓 袁秀芳 冉多良 徐丽华 牛登元 王朝文 王一成 |
| 作者单位： | 1. 新疆农业大学,乌鲁木齐,830052;浙江省农业科学院畜牧兽医研究所,杭州,310021 2. 浙江省农业科学院畜牧兽医研究所,杭州,310021 3. 新疆农业大学,乌鲁木齐,830052 4. 甘肃农业大学,兰州,730070 |
| 基金项目： | 浙江省科技计划重点项目 |
| 摘 要： | 根据GenBank中公布的猪流行性腹泻病毒Ⅳ基因序列，设计了一对特异性引物和探针，扩增长度为186bp的片段。以克隆Ⅳ基因的质粒作为阳性标准品，建立了一种快速检测猪流行腹泻病毒含量的TaqMan荧光定量PCR方法。该方法在10^9-10^1copies／μL范围内具有良好的线性关系，可检测到初始模板中10copies／μL的质粒DNA，以猪圆环病毒、猪乙脑病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病毒和猪细小病毒为模板时，均检测不到荧光信号，而重复性实验中其变异系数均小于2％，表明此方法具有很好的特异性和重复性。应用此方法对采集的60份，陆床样品进行检测，其阳性检出率为92％，而用常规RT-PCR方法进行检测，阳性检出率仅为80％。表明荧光定量RT-PCR方法的敏感性明显高于常规PcR检测方法。 |
| 关 键 词： | 猪流行性腹泻病毒 荧光定量RT-PCR TaqMan荧光探针 |
DEVELOPMENT AND PRELIMINARY APPLICATION OF A TAQMAN-BASED REAL-TIME PCR FOR DETECTION OF PORCINE EPIDEMIC DIARRHEA VIRUS |
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| LIU Deng,YUAN Xiu-fang,RAN Duo-liang,XU Li-hua,NIU Deng-yuan,WANG Chao-wen,WANG Yi-cheng.DEVELOPMENT AND PRELIMINARY APPLICATION OF A TAQMAN-BASED REAL-TIME PCR FOR DETECTION OF PORCINE EPIDEMIC DIARRHEA VIRUS[J].Chinese Journal of Veterinary Parasitology,2010,18(1):28-34. | |
| Authors: | LIU Deng YUAN Xiu-fang RAN Duo-liang XU Li-hua NIU Deng-yuan WANG Chao-wen WANG Yi-cheng |
| Institution: | LIU Deng~(1,2),YUAN Xiu-fang~2,RAN Duo-liang~1,XU Li-hua~2,NIU Deng-yuan~3,WANG Chao-wen~3,WANG Yi-cheng~2 (1.College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China,2.Animal Husb,ry , Veterinary Institute,Zhejiang Academy of Agricultural Science,Hangzhou 310021,3 College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China) |
| Abstract: | A 186 bp fragment was amplified using the specific primers and TaqMan probe designed according to PEDV-N gene sequence deposited in GenBank. Subsequently, a real-time quantitative PCR for detection of PEDV was developed with standard plasmid DNA from the cloned PEDV-N gene. The sensitivity of the assay was 10 copies/μt L plasmid DNA The assay was specific for PEDV because no amplification was found for PCV, JEV, SFV, PRRSV, PRV and PPV. The coefficient variation （CV） of intra/inter-assay for the same DNA sample was less than 2%. The results indicated that the standard curves were shown to be of high linearity, specificity, sensitivity and reproducibility. |
| Keywords: | Porcine epidemic diarrhea virus(PEDV) real-time fluorescent quantitative RT-PCR TaqMan probe |
| 本文献已被 CNKI 维普 万方数据 等数据库收录！ | |