| 鸭痘病毒TaqMan荧光定量PCR检测方法的建立 |
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| 引用本文： | 曹慧慧,孙文超,郑敏,韦显凯,苏姣秀,梁晟,郑列丰,李军,刘棋.鸭痘病毒TaqMan荧光定量PCR检测方法的建立[J].中国畜牧兽医,2015,42(10):2560-2566. |
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| 作者姓名： | 曹慧慧 孙文超 郑敏 韦显凯 苏姣秀 梁晟 郑列丰 李军 刘棋 |
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| 作者单位： | 1. 广西大学动物科学技术学院, 南宁 530001;2. 广西动物疫病预防控制中心, 南宁 530001 |
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| 基金项目： | 国家自然科学基金(31360601);广西科学基金(2012GXNSFAA053073、2013GXNSFBB053006);广西水产畜牧兽医局科技项目(GYMK1204936) |
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| 摘 要： | 为建立检测鸭痘病毒的TaqMan荧光定量PCR方法,本试验克隆了鸭痘病毒P4b基因,构建重组质粒pMD-DPV-P4b,并将其作为标准阳性模板。参照GenBank收录的禽痘病毒P4b基因设计合成1对特异性引物及与该引物相匹配的特异探针。以定量的10倍系列稀释的质粒pMD-DPV-P4b为标准品,通过对反应条件进行优化,建立了一种检测鸭痘病毒的TaqMan荧光定量PCR方法。结果显示,该方法与禽流感病毒、鸭黄病毒、鸭肝炎病毒、新城疫病毒、鸭瘟病毒和小鹅瘟病毒等其他水禽病毒,以及山羊痘病毒和鸡痘病毒等其他痘病毒均无交叉反应,特异性好。该方法最低检测限为1.29×102拷贝/μL,比普通PCR检测方法高100倍。组内和组间变异系数均小于2%。结果表明,本试验所建立方法具有灵敏、特异、安全、快速的特点,适用于鸭痘病毒的检测。
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| 关 键 词： | 鸭痘病毒 禽痘病毒 荧光定量PCR TaqMan探针 |
| 收稿时间： | 2015-05-15 |
Establishment of TaqMan Real-time PCR Method for Detection of Duck Poxvirus |
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| CAO Hui-hui,SUN Wen-chao,ZHENG Min,WEI Xian-kai,SU Jiao-xiu,LIANG Sheng,ZHENG Lie-feng,LI Jun,LIU Qi.Establishment of TaqMan Real-time PCR Method for Detection of Duck Poxvirus[J].China Animal Husbandry & Veterinary Medicine,2015,42(10):2560-2566. |
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| Authors: | CAO Hui-hui SUN Wen-chao ZHENG Min WEI Xian-kai SU Jiao-xiu LIANG Sheng ZHENG Lie-feng LI Jun LIU Qi |
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| Institution: | 1. College of Animal Science and Technology, Guangxi University, Nanning 530001, China;2. Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China |
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| Abstract: | To establishment a TaqMan Real-time PCR method for detection of duck poxvirus (DPV),we cloned the P4b gene of DPV.The specific primers and probe were designed according to the nucleotide sequence of avipoxvirus available in GenBank.Recombinant plasmid pMD-DPV-P4b was employed as positive standard template for Real-time PCR.By optimization of reaction conditions,a TaqMan Real-time PCR method for detection of DPV was established.The results of specificity test proved this method had no cross-react with other waterfowl vial agents and poxviruses including avian influenza virus,duck flavivirus,duck hepatitis virus,Newcastle disease virus,duck entertitis virus,goose parvovirus,goatpox virus and fowlpox virus.The detection limit of the assay was 1.29×102 copies/μL of viral DNA,which was 100 times higher than that of the routine PCR.Reproducibility test showed that the CVs of intra assay and inter assay were both less than 2%.Above results supported that the assay was suitable for the detection of DPV very well. |
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| Keywords: | duck poxvirus avipoxvirus Real-time PCR TaqMan probe |
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