| 非洲猪瘟病毒锁核酸探针荧光定量PCR检测方法的建立 |
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| 引用本文： | 李艳,郭怡德,勾红潮,卞志标,孙铭飞,蔡汝健,宋帅,蒋智勇,楚品品,徐民生,杨东霞,李春玲.非洲猪瘟病毒锁核酸探针荧光定量PCR检测方法的建立[J].中国兽医学报,2021(2):208-212,230. |
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| 作者姓名： | 李艳 郭怡德 勾红潮 卞志标 孙铭飞 蔡汝健 宋帅 蒋智勇 楚品品 徐民生 杨东霞 李春玲 |
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| 作者单位： | ;1.广东省农业科学院动物卫生研究所;2.广东省畜禽疫病防治研究重点实验室;3.农业农村部兽用药物与诊断技术广东科学观测实验站 |
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| 基金项目： | 广东省自然科学基金资助项目(2020A1515010475);广东省重点领域研发计划资助项目(2019B020211005,2019B020217002);国家重点研发计划资助项目(2016YFD0500709)。 |
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| 摘 要： | 根据非洲猪瘟病毒(African swine fever virus,ASFV)P72基因核苷酸序列设计特异性引物和锁核酸(locked nucleic acid,LNA)-TaqMan探针,建立了基于P72基因的LNA-TaqMan探针的ASFV荧光定量PCR方法。结果显示,所建立的LNA-TaqMan探针荧光定量PCR方法具有较高的灵敏度,最低检测限为3.9拷贝/μL,且与猪瘟病毒、猪繁殖与呼吸综合征病毒及猪圆环病毒2型等多种病原不存在交叉反应;该方法的重复性良好,批内和批间变异系数均小于1%;56份临床样品的检测结果与OIE推荐的qPCR方法检测结果一致,符合率为100%。结果表明,本试验所建立的ASFV LNA-TaqMan探针荧光定量PCR方法敏感性、特异性和重复性良好,为ASFV检测提供了一种新的技术选择。
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| 关 键 词： | 非洲猪瘟病毒 P72基因 LNA-TaqMan探针荧光定量PCR |
Establishment of LNA-TaqMan probe real-time PCR for detection of African swine fevervirus |
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| LI Yan,GUO Yide,GOU Hongchao,BIAN Zhibiao,SUN Mingfei,CAI Ru-jian,SONG Shuai,JIANG Zhiyong,CHU Pinpin,XU Minsheng,YANG Dongxia,LI Chunling.Establishment of LNA-TaqMan probe real-time PCR for detection of African swine fevervirus[J].Chinese Journal of Veterinary Science,2021(2):208-212,230. |
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| Authors: | LI Yan GUO Yide GOU Hongchao BIAN Zhibiao SUN Mingfei CAI Ru-jian SONG Shuai JIANG Zhiyong CHU Pinpin XU Minsheng YANG Dongxia LI Chunling |
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| Institution: | (Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;Key Laboratory of Livestock Disease Prevention of Guangdong Province,Guangzhou 510640,China;Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province,Ministry of Agriculture,Guangzhou 510640,China) |
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| Abstract: | In this study,LNA-TaqMan probe-based real-time PCR was established using specific primers and probes according to the P72 gene of African swine fever virus(ASFV).The results showed that the LNA-TaqMan probe-based real-time PCR had higher sensitivity,and the minimum detection limit was 3.9 copies/μL.The LNA-TaqMan probe-based real-time PCR was no cross-reaction with various pathogens such as swine fever virus,porcine reproductive and respiratory syndrome virus,and porcine circovirus type 2.The intra-and inter-assay coefficient of variation was less than 1%,with good repeatability.The results of 56 clinical samples were consistent with the results of qPCR recommended by OIE,and the coincidence rate was 100%.The LNA-TaqMan probe-based real-time PCR established in this study had ideal sensitivity,specificity,and repeatability,which provides a new technology choice for the detection of ASFV. |
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| Keywords: | ASFV P72 gene LNA-TaqMan real-time PCR |
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