本文關(guān)鍵詞： 羊傳染性膿皰病毒 F1L基因 實(shí)時(shí)熒光定量PCR SYBR Green I TaqMan探針　出處：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：羊傳染性膿皰是由羊傳染性膿皰病毒引起的山羊和綿羊的一種急性、接觸性傳染病。為了更快、更準(zhǔn)確的診斷該病,本研究以羊傳染性膿皰病毒F1L基因作為特異性基因,建立了兩種羊傳染性膿皰病毒實(shí)時(shí)熒光定量PCR檢測(cè)方法,即：SYBR Green Ⅰ實(shí)時(shí)熒光定量PCR檢測(cè)方法和TaqMan實(shí)時(shí)熒光定量PCR檢測(cè)方法。根據(jù)GenBank已經(jīng)發(fā)表的CEV參考毒株F1L基因序列,利用Primer Express 3.0軟件針對(duì)兩種方法設(shè)計(jì)合成兩對(duì)特異性引物和一條TaqMan探針。分別構(gòu)建兩種方法的重組陽性質(zhì)粒,作為標(biāo)準(zhǔn)品模板。優(yōu)化兩種方法的反應(yīng)體系和反應(yīng)參數(shù),分別繪制標(biāo)準(zhǔn)曲線,評(píng)價(jià)標(biāo)準(zhǔn)品的相關(guān)性和擴(kuò)增效率,檢驗(yàn)所建立的兩種實(shí)時(shí)熒光定量PCR反應(yīng)體系的重復(fù)性、特異性和敏感性。對(duì)11例羊傳染性膿皰陽性病例病料進(jìn)行了檢測(cè),驗(yàn)證所建立的兩種方法的實(shí)用性。結(jié)果顯示：SYBR GreenⅠ實(shí)時(shí)熒光定量PCR檢測(cè)方法標(biāo)準(zhǔn)曲線的相關(guān)系數(shù)R2=0.99,擴(kuò)增效率E=1.18,最低檢出下限模板濃度為65.47copies/μL,組內(nèi)和組間變異系數(shù)分別為0.17%～0.59%和0.74%-1.25%；TaqMan實(shí)時(shí)熒光定量PCR檢測(cè)方法標(biāo)準(zhǔn)曲線的相關(guān)系數(shù)R2=0.997,擴(kuò)增效率E=1.08,最低檢出下限模板濃度為13.40copies/μL,組內(nèi)和組間變異系數(shù)分別為0.28%-0.55%和0.45%-1.31%。兩種檢測(cè)方法重復(fù)性好,特異性強(qiáng),敏感性高,均與山羊痘活疫苗、口蹄疫O型、亞洲Ⅰ型二價(jià)滅活疫苗及小反芻獸疫病毒疫苗不發(fā)生交叉反應(yīng)；對(duì)收集自不同地區(qū)的11例羊傳染性膿皰病料的檢測(cè)結(jié)果均為陽性。
[Abstract]:Infectious pustules in sheep are an acute, contagious infectious disease in goats and sheep caused by the infectious pustular virus in sheep. In order to diagnose the disease more quickly and accurately. In this study, the F1L gene of sheep infectious pustular virus was used as the specific gene, and two real-time fluorescent quantitative PCR methods were established for the detection of sheep infectious pustular virus. That is: SYBR Green. I Real-time fluorescence quantitative PCR detection method and TaqMan real-time fluorescence quantitative PCR detection method. According to the F1L gene sequence of CEV reference strain published by GenBank. Two pairs of specific primers and one TaqMan probe were designed and synthesized by using Primer Express 3.0 software for the two methods. The recombinant positive plasmids of the two methods were constructed respectively. As standard sample template, optimize the reaction system and parameters of the two methods, draw the standard curve, evaluate the correlation and amplification efficiency of the standard product. The reproducibility, specificity and sensitivity of two real-time fluorescent quantitative PCR reaction systems were tested in 11 sheep infectious pustular positive cases. The results showed that the correlation coefficient of the standard curve of the real-time fluorescence quantitative PCR method for the detection of 1: SYBR Green 鈪，

本文編號(hào)：1472727