本文選題：ASFV + SVDV��； 參考：《四川農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：ASFV、SVDV、FMDV-O、PRV是豬病的4種重要病原。目前我國雖無ASF發(fā)生的報道,但是,由于鄰國俄羅斯多次報道該病的發(fā)生和其可能帶來的嚴重后果,能提早建立一種檢測方法對預(yù)防該病非常必要,且能為將來研究該病提供一定的試驗基礎(chǔ)。SVD、FMD在臨床表現(xiàn)上非常相似,很難從臨床癥狀將它們區(qū)別開來,所以能建立一種快速、準(zhǔn)確檢測和鑒別這兩種病的方法對將來的治療和預(yù)后意義重大。另外,在本實驗室的臨床調(diào)查中,PR一直是近幾年豬群當(dāng)中的高發(fā)病,其惡劣的臨床表現(xiàn)嚴重影響?zhàn)B豬業(yè)的發(fā)展,給畜牧養(yǎng)殖業(yè)帶來了巨大的經(jīng)濟損失。本研究選定了4個靶基因片段,用pMD19-T克隆獲得了T/ASFV、T/ FMDV-O、T/PRV、T/SVDV四個陽性重組質(zhì)粒。在分析了ASFV、SVDV、FMDV-O、PRV致病因子基因的基礎(chǔ)上,依據(jù)靶序列富集多重PCR(Tem-PCR)的原理,設(shè)計了4對特異性的套式PCR引物和探針,并在各內(nèi)引物的5’端分別加上能被一對通用引物識別的標(biāo)簽序列。本試驗建立了一種能同步檢測4種常見病原的Tem-PCR方法。優(yōu)化后的Tem-PCR方法Primer Mix的最佳濃度0.2μM,反應(yīng)的最佳退火溫度為50℃。實驗結(jié)果表明：ASFV、FMDV-O、PRV這3種病原,使用該方法可以在1支反應(yīng)管內(nèi)快速、同步進行檢測,得到大小分別為272bp (ASFV), 576bp (PRV) 233bp(FMDV-O)的特異性產(chǎn)物。Tem-PCR方法對3種病原基因組的檢測靈敏度分別為：3.5×106 copies/μL (ASFV)、4.2×106copies/μL (FMDV-O)、4.6×103 copies/μL (PRV)。特異性試驗發(fā)現(xiàn)該Tem-PCR方法從陽性重組質(zhì)粒T/ASFV、T/FMDV-O、T/PRV基因組中均得到了大量的特異性擴增產(chǎn)物,其他基因組DNA無特異性條帶出現(xiàn)。用優(yōu)化的Tem-PCR方法擴增T/ASFV、T/FMDV-O、T/PRV、T/SVDV,并將產(chǎn)物用基因芯片技術(shù)檢測,結(jié)果所有的雜交質(zhì)控位點及檢測樣品均發(fā)出綠色熒光,所有的陰性質(zhì)控位點均未發(fā)出熒光,表明雜交過程是有效可靠的。
[Abstract]:ASFVV SVDVV FMDV-OFPRV is one of the four important pathogens of swine disease. Although there is no reported occurrence of ASF in our country at present, it is necessary to establish an early detection method for the disease because of the repeated reports on the occurrence of the disease and the serious consequences it may bring in neighboring Russia. And can provide a certain experimental basis for the future study of the disease. SVD FMD in clinical manifestations are very similar, it is difficult to distinguish them from the clinical symptoms, so can establish a rapid, Accurate detection and differential diagnosis of these two diseases are of great significance for future treatment and prognosis. In addition, PR has been a high incidence disease among pigs in our laboratory in recent years. Its poor clinical performance has seriously affected the development of pig industry and brought huge economic losses to livestock industry. In this study, four target gene fragments were selected and four positive recombinant plasmids of T / ASFV / FMDV / T / PRV / T / SVDV were obtained by pMD19-T cloning. Four pairs of specific nested PCR primers and probes were designed on the basis of the analysis of the gene of FMDV-OG PRV pathogenetic factor of SVDVV of ASFV, based on the principle of multiple PCRV-Tem-PCRs enriched by target sequence, four pairs of specific nested PCR primers and probes were designed. At the 5 'end of each internal primer, a label sequence that can be recognized by a pair of universal primers was added. A Tem-PCR method for simultaneous detection of four common pathogens was established. The optimum concentration of Primer Mix is 0.2 渭 m and the optimal annealing temperature is 50 鈩，

本文編號：1841294