非洲猪瘟病毒微滴数字PCR检测方法的建立

详细信息    查看全文 | 推荐本文 |

英文篇名：Development of droplet digital PCR for detection of African swine fever virus 作者：原霖 ; 董浩 ; 倪建强 ; 刘洋 ; 陈亚娜 ; 杨林 ; 宋晓晖 ; 王传彬 英文作者：YUAN Lin;DONGHao;NING Jianqiang;LIU Yang;CHEN Yana;YANG Lin;SONG Xiaohui;WANG Chuanbin;China Animal Disease Control Center; 关键词：非洲猪瘟 ; 微滴数字PCR ; 荧光定量PCR 英文关键词：African Swine Fever;;droplet digital PCR;;real-time quantitative PCR 中文刊名：XMYS 英文刊名：Animal Husbandry & Veterinary Medicine 机构：中国动物疫病预防控制中心; 出版日期：2019-07-10 出版单位：畜牧与兽医 年：2019 期：v.51;No.408 基金：现代农业产业技术体系北京市创新团队(BAIC02-2017) 语种：中文; 页：XMYS201907017 页数：4 CN：07 ISSN：32-1192/S 分类号：87-90

摘要

建立一种具有高灵敏度的检测非洲猪瘟病毒的微滴数字PCR方法,为非洲猪瘟的快速诊断和流行病学研究提供技术支持。在《OIE陆生动物诊断与疫苗手册》中提供的qPCR检测方法的基础上,建立了非洲猪瘟病毒微滴数字PCR方法,对该方法的敏感性、特异性和重复性进行了评估。结果显示,该方法的最低检测限可达0.8 copies/μL,敏感性高于qPCR方法,不与猪常见的6种病毒发生交叉反应,而且重复性较好。采用建立的微滴数字PCR和OIE的qPCR方法分别对78份临床病料进行非洲猪瘟病毒的检测,结果显示2种方法的符合率为100%。本研究建立的微滴数字PCR方法具有理想的敏感性和特异性,适用于临床上非洲猪瘟病毒的检测。
        In this study, a high-sensitivity droplet digital PCR method was established in order to provide technical support for rapid diagnosis and epidemiological study of the African Swine Fever. Based on the qPCR method provided in the OIE Terrestrial Animal Diagnostic and Vaccine Manual, the droplet digital PCR method for African Swine Fever virus detection was established. The limit of detection, specificity and repeatability of this method were evaluated, and the results indicated that its limit of detection was 0.8 copy/μL and its sensitivity was higher than that of the OIE qPCR method. In addition, no cross-reaction was observed when the method was assayed with 6 common swine viruses, and the repeatability of the droplet digital PCR was also good. 78 clinical samples were detected by the established droplet digital PCR and the OIE qPCR, respectively. The result showed that the coincidence rate of the two methods was 100%. The droplet digital PCR method established in this study had ideal sensitivity and specificity, and was suitable for the detection of African Swine Fever virus in clinical practice.

引文

[1] 国家中长期动物疫病防治规划(2012-2020年)[J].黑龙江畜牧兽医,2012(18):26-30.
    [2] 张志,康京丽,李晓成.非洲猪瘟的流行特征和传播路线[J].中国动物检疫,2018,35(11):48-51.
    [3] Pinheiro L B,Coleman V A,Hindson C M,et al.Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification[J].Analytical Chemistry,2012,84(2):1003-1011.
    [4] 林佳琪,苏国成,苏文金,等.数字PCR技术及应用研究进展[J].生物工程学报,2017,33(2):170-177.
    [5] 邬旭龙,肖璐,宋勇,等.非洲猪瘟病毒微滴数字PCR(ddPCR)方法的建立及应用[J].微生物学通报,2017,44(12):2839-2846.