马动脉炎病毒核酸与蛋白定量检测方法的建立
发布时间：2019-01-07 22:39
【摘要】：马病毒性动脉炎(Equine viral arteritis,EVA)是由马动脉炎病毒(Equine arteritis virus,EAV)引起的一种马属动物的传染病。为了建立能够快速、定量检测EAV的病原学方法,本研究应用两株抗EAV N蛋白的单克隆抗体(2B3和2B9)建立了在蛋白水平定量检测EAV的抗原捕捉ELISA (AC-ELISA)方法。结果表明,该方法对EAV的最低检出限为3.86 TCID50,特异性良好,与马属动物常见病毒不发生交叉反应,3次重复试验测定表明该方法稳定性高。同时本研究通过对EAV (Bucyrus株)基因组ORF7设计一对特异性引物,应用新型染料Eva Green建立了能够在核酸水平定量检测EAV的RT-PCR方法,结果表明,当病毒载量在102～107拷贝/μL时,应用该方法测得的Ct值与EAV病毒含量具有良好的线性关系,该方法能够检测出的最低EAV滴度为101.5TCID50/mL,对标准质粒的检出限为10拷贝/μL,经三次组内和组间重复试验测定,106、104、102拷贝/μL的标准质粒测得的Ct值的变异系数全部小于2%,表明该方法重复性良好。本研究建立的这两种检测方法为实验室开展EAV病原学研究奠定了良好的实验基础。
[Abstract]:Equine viral arteritis (Equine viral arteritis,EVA) is an infectious disease caused by equine arteritis virus (Equine arteritis virus,EAV). In order to establish a rapid and quantitative method for the detection of EAV, two monoclonal antibodies (2B3 and 2B9) against EAV N protein were used to establish an antigen capture ELISA (AC-ELISA) method for quantitative detection of EAV at protein level. The results showed that the minimum detection limit for EAV was 3.86 TCID50, and the method did not cross react with the common viruses in equine. The stability of the method was proved to be high by three repeated tests. At the same time, a pair of specific primers were designed for genomic ORF7 of EAV (Bucyrus strain, and a new type of dye Eva Green was used to establish a RT-PCR method for quantitative detection of EAV at nucleic acid level. The results showed that when the viral load was 102107copies / 渭 L, The Ct value obtained by this method has a good linear relationship with the EAV virus content. The lowest EAV titer detected by this method is 101.5 TCID 50% mL, and the detection limit for the standard plasmid is 10 copies / 渭 L. The Ct values measured by 106104102 copies / 渭 L standard plasmids were all less than 2, which indicated that the method had good reproducibility. The two detection methods established in this study laid a good experimental foundation for laboratory research on EAV etiology.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.652
本文编号：2404254
[Abstract]:Equine viral arteritis (Equine viral arteritis,EVA) is an infectious disease caused by equine arteritis virus (Equine arteritis virus,EAV). In order to establish a rapid and quantitative method for the detection of EAV, two monoclonal antibodies (2B3 and 2B9) against EAV N protein were used to establish an antigen capture ELISA (AC-ELISA) method for quantitative detection of EAV at protein level. The results showed that the minimum detection limit for EAV was 3.86 TCID50, and the method did not cross react with the common viruses in equine. The stability of the method was proved to be high by three repeated tests. At the same time, a pair of specific primers were designed for genomic ORF7 of EAV (Bucyrus strain, and a new type of dye Eva Green was used to establish a RT-PCR method for quantitative detection of EAV at nucleic acid level. The results showed that when the viral load was 102107copies / 渭 L, The Ct value obtained by this method has a good linear relationship with the EAV virus content. The lowest EAV titer detected by this method is 101.5 TCID 50% mL, and the detection limit for the standard plasmid is 10 copies / 渭 L. The Ct values measured by 106104102 copies / 渭 L standard plasmids were all less than 2, which indicated that the method had good reproducibility. The two detection methods established in this study laid a good experimental foundation for laboratory research on EAV etiology.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.652
【参考文献】
相关期刊论文 前4条
1 黄正才;马的病毒性动脉炎[J];四川畜牧兽医;1997年04期
2 梁成珠;曹瑞兵;魏建超;朱来华;陈溥言;高宏伟;;马动脉炎病毒GL蛋白主要抗原域的表达及间接ELISA的初步建立[J];微生物学报;2006年03期
3 洪健;周雪平;;ICTV第八次报告的最新病毒分类系统[J];Virologica Sinica;2006年01期
4 胡月;戚亭;王晓钧;;马病毒性动脉炎[J];畜牧兽医科技信息;2013年10期
相关硕士学位论文 前1条
1 赵世华;抗马动脉炎病毒病毒N蛋白单克隆抗体的制备及表位鉴定和固相竞争ELISA方法的建立[D];中国农业科学院;2013年
，本文编号：2404254
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2404254.html
