ASFV、SVDV、FMDV-O、PRV的TEM-PCR检测方法的初步研究

发布时间：2018-05-04 03:05

  本文选题：ASFV + SVDV　； 参考：《四川农业大学》2015年硕士论文

【摘要】：ASFV、SVDV、FMDV-O、PRV是猪病的4种重要病原。目前我国虽无ASF发生的报道,但是,由于邻国俄罗斯多次报道该病的发生和其可能带来的严重后果,能提早建立一种检测方法对预防该病非常必要,且能为将来研究该病提供一定的试验基础。SVD、FMD在临床表现上非常相似,很难从临床症状将它们区别开来,所以能建立一种快速、准确检测和鉴别这两种病的方法对将来的治疗和预后意义重大。另外,在本实验室的临床调查中,PR一直是近几年猪群当中的高发病,其恶劣的临床表现严重影响养猪业的发展,给畜牧养殖业带来了巨大的经济损失。本研究选定了4个靶基因片段,用pMD19-T克隆获得了T/ASFV、T/ FMDV-O、T/PRV、T/SVDV四个阳性重组质粒。在分析了ASFV、SVDV、FMDV-O、PRV致病因子基因的基础上,依据靶序列富集多重PCR(Tem-PCR)的原理,设计了4对特异性的套式PCR引物和探针,并在各内引物的5’端分别加上能被一对通用引物识别的标签序列。本试验建立了一种能同步检测4种常见病原的Tem-PCR方法。优化后的Tem-PCR方法Primer Mix的最佳浓度0.2μM,反应的最佳退火温度为50℃。实验结果表明：ASFV、FMDV-O、PRV这3种病原,使用该方法可以在1支反应管内快速、同步进行检测,得到大小分别为272bp (ASFV), 576bp (PRV) 233bp(FMDV-O)的特异性产物。Tem-PCR方法对3种病原基因组的检测灵敏度分别为：3.5×106 copies/μL (ASFV)、4.2×106copies/μL (FMDV-O)、4.6×103 copies/μL (PRV)。特异性试验发现该Tem-PCR方法从阳性重组质粒T/ASFV、T/FMDV-O、T/PRV基因组中均得到了大量的特异性扩增产物,其他基因组DNA无特异性条带出现。用优化的Tem-PCR方法扩增T/ASFV、T/FMDV-O、T/PRV、T/SVDV,并将产物用基因芯片技术检测,结果所有的杂交质控位点及检测样品均发出绿色荧光,所有的阴性质控位点均未发出荧光,表明杂交过程是有效可靠的。
[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，

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