猪传染性胃肠炎病毒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 porcine transmissible gastroenteritis virus
张羽欣 1王树茂 2段宏勇 2赵款 3董世山 3高飞 4李丽薇4
作者信息
- 1. 河北农业大学动物医学院,河北保定 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
- 3. 河北农业大学动物医学院,河北保定 071001
- 4. 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,江苏扬州 225009
- 折叠
摘要
本研究针对猪传染性胃肠炎病毒(TGEV)的N基因保守序列设计特异性引物和探针,构建重组质粒,建立针对TGEV N基因的TaqMan荧光定量PCR检测方法,用于临床快速诊断和实验室检测.结果显示,该方法特异性好,与其他具有相似临床症状的猪源病毒相比,不存在交叉反应,Ct值在1×109～1× 101 copies/μL的范围内具有很好的线性关系,R2=0.99.该方法最低检测限为10 copies/μL,标准曲线为y=-3.058 7x+37.7,斜率为-3.058 7.该方法重复性好、敏感度高、特异性强,适用于临床样品中TGEV抗原核酸的检测和实验室病毒生长特性的研究,为准确定量及诊断TGEV提供了有效的试验手段和检测工具.
Abstract
In this study,we designed highly specific primers and probes for the N gene fragment of porcine transmissible gastroenteritis virus(TGEV),constructed the recombinant plasmid,and estab-lished the TaqMan fluorescence quantitative PCR detection method for the rapid clinical diagnosis and laboratory testing.The results showed that the method had good specificity.Compared with other porcine viruses,there was no cross-reaction between viruses with similar clinical symptoms.The Ct val-ues displayed good linear relationship in the range of 1×n109-1 × 101 copies/μL,R2=0.99.The lowest detection limit of this method was 10 copies/μL.The standard curve was y=-3.058 7x+37.7,and the slope was-3.058 7.The method has good repeatability,high sensibility,and strong specificity.The method is suitable for the detection of TGEV antigen nucleic acid in clinical samples and the study of the growth characteristics of laboratory viruses.It provides an effective test and detection tool for the accurate quantification and diagnosis of TGEV.
关键词
猪传染性胃肠炎病毒/N基因/荧光定量PCR/TaqManKey words
transmissible gastroenteritis virus/Ngene/fluorescent quantitative PCR/TaqMan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)
国家重点研发计划(2022YFD1800305)
国家重点研发计划(2022YFD1800804)
国家重点研发计划(2021YFD1801401)
中国农科院科技创新工程项目(CAAS-CSLPDCP-202301)
出版年
2024
国家科技期刊平台
NSTL
万方数据