针对非洲猪瘟病毒K205R基因实时荧光定量PCR检测方法的建立与应用
Development and Application of TaqMan Real-Time Quantitative PCR for Detection of African Swine Fever Virus K205R Gene
乔思娜 1李丽薇 2赵款 3高飞 2周艳君 2姜一峰 2童武 2赵冉 4童光志 2李国新 2董世山3
作者信息
- 1. 河北农业大学动物医学院,保定 071001;中国农业科学院上海兽医研究所,上海 200241
- 2. 中国农业科学院上海兽医研究所,上海 200241;江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州 225009
- 3. 河北农业大学动物医学院,保定 071001
- 4. 厦门市动物疫病预防控制中心,厦门 361009
- 折叠
摘要
为了快速、准确检测非洲猪瘟病毒(African swin fever virus,ASFV),本文根据我国流行毒株ASFV SY18(GenBank ID:MH766894.1)已公布的K205R基因序列设计并合成特异性引物及探针,建立针对K205R基因的TaqMan荧光定量PCR检测方法.经过特异性试验、敏感性试验、重复性试验和临床样品检测试验分析显示,本文建立的TaqMan荧光定量PCR检测方法Ct值与标准品在1×1011～1×102 copies/μL范围内具有很好的线性关系,相关系数为0.998,斜率为-2.797,检测下限为102 copies/μL,与其他能引起相似临床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1.53774%,重复性好.本方法适用于临床样品及本实验室构建的表达ASFV K205R基因的重组猪繁殖与呼吸综合征病毒的鉴定及检测,对ASFV快速检测及ASF早期诊断有重要意义.
Abstract
In order to rapidly and accurately detect African swine fever virus(ASFV),this study designed and synthesized primers and probes based on the published K205R gene sequence of the prevalent strain ASFV SY18(GenBank ID:MH766894.1)in China and developed a TaqMan fluorescent quantitative PCR assay.The Ct value of the assay showed a good linear relationship with the standard in the range of 1×1011 to 1×102 copies/μL with a correlation coefficient of 0.998 and a slope of-2.797.The lower limit of detection was 102 copies/μL and there was no cross-reactivity with other porcine viruses that caused similar signs.In addition,the coefficients of variation between and within groups was less than 1.53774%.In summary,the TaqMan fluorescent quantitative PCR assay developed in this study had good specificity,sensitivity and reproducibility,which could be used for early diagnosis of ASF and rapid detection of ASFV and recombinant porcine reproductive and respiratory syndrome virus expressing ASFV K205R protein.
关键词
非洲猪瘟病毒(ASFV)/K205R基因/荧光定量PCR/检测方法Key words
African swine fever virus/K205R gene/real-time quantitative PCR/detection引用本文复制引用
基金项目
上海市科技兴农项目(2022-02-08-00-12-F01162)
河北省省属高等学校基本科研业务费(KY202017)
广东省重点领域研发计划资助(2019B020211003)
河北省重点研发计划项目(20326622D)
出版年
2024
国家科技期刊平台
NSTL
万方数据