| 非洲猪瘟病毒TaqMan荧光定量PCR检测方法的建立 |
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| 引用本文： | 吴亚楠,朱潇静,周博伦,张博,刘晴晴,郑兰兰,魏战勇.非洲猪瘟病毒TaqMan荧光定量PCR检测方法的建立[J].中国兽医学报,2020(5):888-891,896. |
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| 作者姓名： | 吴亚楠 朱潇静 周博伦 张博 刘晴晴 郑兰兰 魏战勇 |
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| 作者单位： | 河南农业大学牧医工程学院;河南省动物性食品安全重点实验室 |
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| 基金项目： | 国家重点研发计划资助项目(2018YFD0501205);许昌市基础与前沿资助项目(JC2018005)。 |
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| 摘 要： | 建立一种TaqMan荧光定量PCR检测方法,用于非洲猪瘟病毒(African swine fever virus,ASFV)保守结构蛋白P72基因的检测。参照GenBank登录的ASFV P72相关基因序列,设计合成1对特异性引物与1个TaqMan探针,通过对反应条件和反应体系进行优化,建立了快速检测ASFV的TaqMan实时荧光定量PCR方法,对该方法的灵敏性、特异性与重复性进行了验证。结果显示,该方法能有效扩增1.0×10^0~1.0×10^9拷贝/μL ASFV-P72标准质粒,建立的标准曲线呈现良好的线性关系;检测灵敏度为1.0×10^0拷贝;对猪流行性腹泻病毒、猪δ冠状病毒、高致病性猪繁殖与呼吸综合征病毒、猪伪狂犬病毒等病原不发生交叉反应;重复性试验结果显示变异系数(CV)小于1%。结果表明,本试验建立的TaqMan荧光定量PCR检测方法具有良好的灵敏性、特异性和重复性,可用于临床样本中ASFV的检测,从而更好地对ASF疫情进行监测和诊断。
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| 关 键 词： | 非洲猪瘟病毒 P72基因 TaqMan实时荧光定量PCR |
Development of a TaqMan real-time fluorescent RT-PCR for specific detection of African swine fever virus |
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| WU Ya-nan,ZHU Xiao-jing,ZHOU Bo-lun,ZHANG Bo,LIU Qing-qing,ZHENG Lan-lan,WEI Zhan-yong.Development of a TaqMan real-time fluorescent RT-PCR for specific detection of African swine fever virus[J].Chinese Journal of Veterinary Science,2020(5):888-891,896. |
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| Authors: | WU Ya-nan ZHU Xiao-jing ZHOU Bo-lun ZHANG Bo LIU Qing-qing ZHENG Lan-lan WEI Zhan-yong |
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| Institution: | (College of Animal Husbandry and Veterinary,Henan Agricultural University,Zhengzhou 450002,China;Henan Animal Food Safety Key Laboratory,Zhengzhou 450002,China) |
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| Abstract: | This experiment was conducted to develop a TaqMan real-time PCR method for detection of African swine fever virus(ASFV).A pairs of specific primers and a TaqMan probe were designed and synthesized according to the ASFV P72 gene sequence registered in GenBank.Then a real-time fluorescent RT-PCR assay using the TaqMan probe was developed by optimizing the conditions and systems of reaction.Sensitivity,specificity and reproducibility of the method were also determined.The results showed that the TaqMan real-time PCR could detect the ASFV between 1.0×10^0-1.0×10^9 copies/μLwith good linear relation,and the sensitivity limit was 1.0×10^0 copies/μL.And the nucleic acid of porcine epidemic diarrhea virus(PEDV),porcine deltacoronavirus(PDCoV),highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV),pseudorabies virus(PRV) and other porcine viruses could not be detected using this TaqMan real-time PCR method,which indicated the high specifity of the method.The CV(coefficient of variation) of intra-assay and inter-assay using the TaqMan real-time PCR were no more than 1%.The TaqMan real-time PCR method established here has good sensitivity,specificity and reproducibility,which can be used for the ASFV detection in clinical samples. |
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| Keywords: | African swine fever virus(ASFV) P72 real-time fluorescent quantitative PCR |
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