【作者】 蔡阳；

【导师】 钱爱东；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 本研究根据已发表的猪圆环病毒PCV-1和PCV-2的全基因序列，利用DNAstar软件对PCV-1和PCV-2的全基因序列进行同源性比较，应用Oligo6.0软件设计出两对引物，其中一对PCV特异性引物P1和P2，扩增出PCV-1和PCV-2上长938bp的片段；另一对为PCV-2型特异性引物P3和P4，扩增片段长490bp。复合PCR方法可以检测到1ng的病毒核酸样品。 建立了定量检测猪圆环病毒DNA的竞争PCR方法。PCR扩增PCV-2 ORF2上490bp片段P3P4，构建了目标模板。将P3P4片段应用BanⅡ酶切，再进行连接，之后克隆到pMD18-T载体，构建含694bp的c-P3p4 DNA片段的竞争模板，其携带同目标DNA和样本DNA相同的引物结合区。以定量的竞争模板同一系列稀释的竞争模板进行竞争PCR扩增，结果当反应体系中起始模板的量为1.0×10~7copies／ml，循环次数小于或等于30时，原始模板数以指数增长。以获得目标模板浓度和扩增产物的荧光强度(IOD)比值的回归曲线，将该曲线作为标准曲线。根据竞争模板和目标模板产物之间的荧光强度(IOD)差异，引进了校正系数(Lc／Lt)。将反应中的各种参数带回回归方程，获得根据样品量和PCR产物荧光强度计算浓度的公式。初步应用竞争定量PCR方法检测感染细胞内的PCV-2基因组的拷贝数增殖变化情况和临床样品中的PCV-2 DNA含量，这为猪圆环病毒核酸诊断和疫苗研究奠定了基础。更多还原

【Abstract】 A multiplex polymerase chain reaction was developed to simultamneous detect type 1 and type 2 of porcine circovirus. The homology of the full sequences of PCV reported and registered in Genebank of different strains of PCV-1 and PCV-2 was respectively and compared with each other. Two sets of specific primers of PCV were designed. PCV can be amplified 938bp, PCV-2 can be amplified 490bp.The designed primers were used to PCR reaction. The result showed that the design of the two sets of primers was right. Then the optimal condition of PCR for PCV were determined.Competitive quantitative PCR (cPCR) assay was developed for monitoring and detecting porcine circovirus (PCV-2) DNA. Using PCR assay, the cPCR was based on competitive co-amplification of a 490-bp region of the PCV-2 ORF2 gene, with a known concentration of competitor DNA, which produced a 692-bp fragment. The cPCR was validated by quantification of a known amount of competitor PCV DNA with one series of dilutions of target DNA. Using the regression equation, we protracted the standard curve and affirmed the quantity of the competitive template. According to the different of the IOD between the products of competitor and target, introduce Lc/Lt as the adjust coefficient. After all parameter regressed, got the concentration formulae that based on the quantity of the samples and the IOD of the PCR products.Used this technique to determine PCV genome copy numbers in infected cells. Furthermore, we measured PCV DNA loads in clinical samples and found PCV-2 DNA content in the piglet with PMWS is higher than 1.0145 x 108copies/ml in lung, 0.8 x 107copies/ml in blood, and 9.698 x 108copies/ml in lymph. Our research might provide some hints to the study of the future work.更多还原