| TaqMan探针实时荧光定量PCR诊断犬瘟热病毒方法的建立 |
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| 作者姓名： | 苏建青 郑学星 候小强 岳妙姝 夏咸柱 |
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| 作者单位： | 吉林大学畜牧兽医学院，解放军军事医学科学院军事兽医研究所 长春130062，河北北方学院动医系，张家口075131，吉林大学畜牧兽医学院，吉林大学畜牧兽医学院，长春理工大学生命科学技术学院，解放军军事医学科学院军事兽医研究所，长春130062，长春130062，长春130062，长春，130062，长春130062 |
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| 基金项目： | 解放军总后勤部科技攻关项目 |
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| 摘 要： | 根据犬瘟热病毒核蛋白基因的保守序列分别设计一对引物及其相应的Taqman探针建立荧光定量PCR。构建FQ-PCR绝对定量的标准品,并对反应体系进行优化,建立绝对定量的标准曲线。利用十倍稀释法检验方法的灵敏度并与普通RT-PCR法进行比较;特异性检验后进行临床标本的检验。结果显示:实验成功构建了绝对定量的参照质粒,标准曲线相关系数为0.994。犬瘟热病毒FQ-PCR检测反应的灵敏度为10TCID50;5种非犬瘟热病毒病原体检测均为阴性;说明实验建立的FQ-PCR具有快速、灵敏和稳定的特点,适用于临床样品的检验。
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| 关 键 词： | TaqMan探针 荧光定量 犬瘟热病毒 |
Establishment of a TaqMan-based Real-Time FQ-PCR Assay for Detection of Canine Distemper Virus |
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| Authors: | Su Jianqing Zheng Xuexing Hou Xiaoqiang Yue Miaoshu Xia Xianzhu |
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| Institution: | 1College of Animal Science and Veterinary Medicine, Jilin University, ChangChun 130062, 2 Institute of Military Veterinary, Academy of Military Medical Sciences of PLA, Changchun 130062, 3Department of Animal Medicine, Hebei Northern College, Zhangjiakou |
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| Abstract: | According to conservative sequence of canine distemper virus nuclear proteins gene,a pair primers and TaqMan probe were designed,respectively.The standard recombinant plasmids for N gene were constructed as reference standard used in absolute quantification assay.The reaction conditions of FQ-PCR were optimized.The sensitivity of assay was tested with ten fold serial dilution of CDV and compared with ordinary RT-PCR.The quantitative curve was established using reference standard plasmids.The clinic samples were detected after specificity assay.The results demonstrated that the plasmid for FQ-PCR was favorable.The regression coefficient of the quantitative curve was 0.994.The detection sensitivity of FQ-PCR was 10 TCID50 and the specificity was determined by testing five other specimens,all of which yielded negative results.The detection system based on real-time RT-PCR was rapid,sensitive and steady,which could be used to detect the clinic samples. |
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| Keywords: | TaqMan probe flurogenic quantitative Canine Distemper Virus |
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