非洲猪瘟TaqMan实时荧光定量PCR检测方法的建立与应用  

Establishment and application of TaqMan real-time quantitative PCR for detection of African swine fever virus

作　　者：杜楠楠 高飞[2,3] 姜一峰 郑浩[2,3] 童武 李国新[2,3] 张宽 张玉娇[2] 黄剑 童光志[2,3] DU Nan-nan;GAO Fei;JIANG Yi-feng;ZHENG Hao;TONG Wu;LI Guo-xin;ZHANG Kuan;ZHANG Yu-jiao;HUANG Jian;TONG Guang-zhi(College of Animal Science.Fujian Agriculture and Forestry University College,Fuzhou 350002,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Jinngsu Co-Innovaiion Center for the Prevention and Control of Important Animal Infcinus Disease and Zoonosis,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]福建农林大学动物科学学院,福建福州350002 [2]中国农业科学院上海兽医研究所,上海200241 [3]扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2020年第4期423-429,共7页Chinese Veterinary Science

基　　金：广东省重点领域研发计划项目:非洲猪瘟应急综合防控技术研究及应用(2019B020211003);广东省畜禽疫病防治研究重点实验室开发基金项目:非洲猪瘟病毒荧光定量快速检测方法的建立及应用(YDWS1906)。

摘　　要：为建立检测非洲猪瘟病毒(African swine fever virus,ASFV)的快速诊断方法,本研究根据ASFV SY18株p17蛋白的编码基因D117L的保守序列设计并合成引物和探针,建立了基于ASFV p17蛋白的编码基因D117L的TaqMan荧光定量PCR检测方法,并验证其特异性、灵敏性和重复性。结果显示,本研究建立的TaqMan荧光定量PCR检测方法的C_t值与标准品在1×10^9~1×10^1copies/μL范围内呈良好的线性关系,相关系数为0.998,斜率为-3.192,检测下限为10 copies/μL,且与其他能引起相似症状的猪源病毒无交叉反应。重复性试验结果显示,组间与组内变异系数均小于1.920 7%,重复性好。此方法可用于ASF的早期诊断和ASFV快速检测。In order to establish a rapid diagnostic method for the detection of African swine fever virus(ASFV).In this study,primers and probes were designed and synthesized according to the conserved sequence of the coding gene D117L of ASFV SY18 strain p17 protein,and Taq Man real-time quantitative PCR detection method for the coding gene D117L of ASFV p17 protein was established.The method has been proved with high sensitivity,high specificity and repeata bility.The test results showed that the Ct value showed a good linear relationship with the standard in the range of 1×10^9~1×10^1 copies/μL and the correlation was 0.998 with a slope of-3.192.The detection limit is 10 copies/μL,and there was no cross-reaction with other porcine viruses that cause similar symptoms.The repeatability test showed that the inter-assay and intra-assay coefficient of variation were both less than 1.9207%,indicating a good repeatability.This method can be used for the early diagnosis of ASF and rapid detection of ASFV in the future.

关 键 词：非洲猪瘟病毒 p17蛋白 D117L基因 实时荧光定量PCR 

分 类 号：S852.659.1[农业科学—基础兽医学]