發(fā)布時(shí)間：2019-01-07 22:39

【摘要】：馬病毒性動(dòng)脈炎(Equine viral arteritis,EVA)是由馬動(dòng)脈炎病毒(Equine arteritis virus,EAV)引起的一種馬屬動(dòng)物的傳染病。為了建立能夠快速、定量檢測(cè)EAV的病原學(xué)方法,本研究應(yīng)用兩株抗EAV N蛋白的單克隆抗體(2B3和2B9)建立了在蛋白水平定量檢測(cè)EAV的抗原捕捉ELISA (AC-ELISA)方法。結(jié)果表明,該方法對(duì)EAV的最低檢出限為3.86 TCID50,特異性良好,與馬屬動(dòng)物常見病毒不發(fā)生交叉反應(yīng),3次重復(fù)試驗(yàn)測(cè)定表明該方法穩(wěn)定性高。同時(shí)本研究通過對(duì)EAV (Bucyrus株)基因組ORF7設(shè)計(jì)一對(duì)特異性引物,應(yīng)用新型染料Eva Green建立了能夠在核酸水平定量檢測(cè)EAV的RT-PCR方法,結(jié)果表明,當(dāng)病毒載量在102～107拷貝/μL時(shí),應(yīng)用該方法測(cè)得的Ct值與EAV病毒含量具有良好的線性關(guān)系,該方法能夠檢測(cè)出的最低EAV滴度為101.5TCID50/mL,對(duì)標(biāo)準(zhǔn)質(zhì)粒的檢出限為10拷貝/μL,經(jīng)三次組內(nèi)和組間重復(fù)試驗(yàn)測(cè)定,106、104、102拷貝/μL的標(biāo)準(zhǔn)質(zhì)粒測(cè)得的Ct值的變異系數(shù)全部小于2%,表明該方法重復(fù)性良好。本研究建立的這兩種檢測(cè)方法為實(shí)驗(yàn)室開展EAV病原學(xué)研究奠定了良好的實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.652

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 黃正才;馬的病毒性動(dòng)脈炎[J];四川畜牧獸醫(yī);1997年04期

2 梁成珠;曹瑞兵;魏建超;朱來華;陳溥言;高宏偉;;馬動(dòng)脈炎病毒GL蛋白主要抗原域的表達(dá)及間接ELISA的初步建立[J];微生物學(xué)報(bào);2006年03期

3 洪健;周雪平;;ICTV第八次報(bào)告的最新病毒分類系統(tǒng)[J];Virologica Sinica;2006年01期

4 胡月;戚亭;王曉鈞;;馬病毒性動(dòng)脈炎[J];畜牧獸醫(yī)科技信息;2013年10期

相關(guān)碩士學(xué)位論文 前1條

1 趙世華;抗馬動(dòng)脈炎病毒病毒N蛋白單克隆抗體的制備及表位鑒定和固相競爭ELISA方法的建立[D];中國農(nóng)業(yè)科學(xué)院;2013年

，

本文編號(hào)：2404254