| 非洲猪瘟病毒TaqMan探针法荧光定量PCR检测方法建立 |
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| 引用本文： | 任名,牛婷婷,于婉琪,孙海伟,邬静,陈鸿军.非洲猪瘟病毒TaqMan探针法荧光定量PCR检测方法建立[J].中国动物传染病学报,2020(3):42-48. |
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| 作者姓名： | 任名 牛婷婷 于婉琪 孙海伟 邬静 陈鸿军 |
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| 作者单位： | 中国农业科学院上海兽医研究所;湖南农业大学动物医学院 |
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| 基金项目： | “十三五”国家重点研发专项“烈性外来动物疫病防控技术研发”资助(2017YFD0502302)。 |
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| 摘 要： | 为建立一种快速、准确的非洲猪瘟病毒(ASFV)分子检测方法,以2018年8月分离的SY18毒株P72基因序列(GenBank登录号:MH713612.1)为参考毒株,人工合成质粒标准品,命名为pcDNA3-P72,基于该毒株P72基因序列,在1627~1876 bp处设计一对特异性引物和TaqMan探针,优化反应体系及条件,本研究成功建立了基于P72基因的TaqMan探针qPCR方法,并与OIE的特异性引物和TaqMan探针进行灵敏度、稳定性和特异性比较.结果显示:本研究建立的qPCR检测方法,标准曲线线性关系良好,R^2值可达到0.9932,灵敏性最低能检测到10 copies/μL,与OIE引物扩增灵敏度相当,比普通PCR方法高100倍.该方法重复性良好,批内和批间变异系数均小于1%.这一方法为我国非洲猪瘟疫情的快速确诊提供了必要的分子诊断工具.
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| 关 键 词： | 非洲猪瘟病毒 TAQMAN 荧光定量PCR 分子诊断 |
DEVELOPMENT OF TAQMAN PROBE REAL-TIME PCR METHOD FOR DETECTION OF AFRICAN SWINE FEVER VIRUS |
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| REN Ming,NIU Ting-ting,YU Wan-qi,SUN Hai-wei,WU Jing,CHEN Hong-jun.DEVELOPMENT OF TAQMAN PROBE REAL-TIME PCR METHOD FOR DETECTION OF AFRICAN SWINE FEVER VIRUS[J].Chinese Journal of Animal Infectious Diseases,2020(3):42-48. |
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| Authors: | REN Ming NIU Ting-ting YU Wan-qi SUN Hai-wei WU Jing CHEN Hong-jun |
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| Institution: | (Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Hunan Agricultural University,Changsha 410128,China) |
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| Abstract: | In the present study,TaqMan probe fluorescence quantitative polymerase chain reaction(qPCR)technique was developed for rapid detection of African swine fever virus(ASFV).The standard plasmid pcDNA3-P72 was constructed based on the P72 gene of the recent Chinese strain SY18(accession no.MH713612.1 in GenBank).In addition,a pair of specific primers and TaqMan probe were designed at 1627-1876 nt of P72 gene sequence.Subsequently,the qPCR method developed here was optimized for its reaction system and conditions.The results showed that the P72 qPCR method had a good linear relationship along with the standard curve.The R^2 value reached to 0.9932,and the detection limit was 10 copies/μL,which was the same as the method followed with the OIE primers but 100 times higher sensitivity than the common PCR method.The intra-and inter-assay coefficients of variation were less than 1%,indicting its good repeatability.The qPCR method developed in the present study might be used for molecular diagnosis of ASFV. |
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| Keywords: | African swine fever virus TaqMan real-time PCR molecular diagnosis |
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