【作者】 高淑霞；

【导师】 庄文忠； 牛钟相；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2006， 博士

【摘要】 混合感染已经成为猪群发病的普遍现象,使病情更为复杂,增加了临床和实验室诊断难度,因此有必要建立一种一次多元的检测方法。基因芯片在病原体检测方面具有独特的优势,它以高通量并行检测的方式,可以使病料中的多种病原体同时被检出,具有快速、高效、灵敏的特点,尤其适宜于大规模的样本检测。本研究采用基因芯片技术,借助多重PCR和核酸标记技术,构建了猪病毒性呼吸道病诊断基因芯片和猪病毒性繁殖障碍病诊断基因芯片,用于混合感染病料的检测,具有同步检测和鉴别诊断的功能。主要研究内容如下:试验一探针的克隆与鉴定根据Genbank收录的PRRSV、CSFV、PCV-2、PPV、PRV、JEV和A型流感病毒核酸序列,针对不同病毒的保守区设计引物。用高保真DNA聚合酶扩增病毒DNA或cDNA,得到了7种病毒的特征性核酸片段,纯化后与载体pMD18-T Simple Vector连接,构建重组质粒。PCR和测序鉴定结果证明,重组质粒构建成功。多序列同源性比较结果显示,探针序列与相关病毒分离株的同源性为89~100%。重组质粒的成功构建为基因芯片探针的制备提供了可靠、稳定的材料来源,保证了芯片检测的特异性。试验二猪病毒性呼吸道病诊断基因芯片的构建和初步应用以试验一构建的重组质粒制备PRRSV、PRV、PCV-2和SIV探针,制备猪病毒性呼吸道病诊断基因芯片。Biotin-11-dUTP使用浓度的选择:在PCR扩增过程用Biotin-11-dUTP替代部分dTTP对靶片段进行标记,扩增体系中Biotin-11-dUTP的终浓度分别为40μM、50μM、70μM和100μM。检测结果显示,4个浓度体系的产物都可以得到杂交信号,而且杂交信号随着浓度的递增而增强。70μM(替代35%的dTTP)的终浓度可以满足检测分析的要求。杂交温度的选择:S、PRRSV、PRV、PCV-2、SIV的靶片段分别在37℃、42℃、50℃和55℃四种不同的杂交温度条件下与芯片杂交。检测结果表明,各探针和阳性对照位点在不同温度条件下都有杂交信号,但多数在42℃条件下获得较高的SNR值。各靶片段在不同温度条件下与其它探针之间无交叉杂交信号。杂交时间的选择:在40min、1h、1.5h和2.0h不同杂交时间内,各靶片段与芯片的杂交结果显示,随着杂交时间的延长杂交信号有增强的趋势。杂交时间为1h时,各探更多还原

【Abstract】 Multiple infections have become widespread in swinery, and have made the conditions more complex. This case increased the difficulty of clinical and laboratory diagnosis. Therefore, it’s necessary to establish a pluralistic method to diagnosis the complex infective diseases. In this study, the diagnostic DNA macroarray for porcine viral disease has been constructed.Major study reads as follows:Part 1: Clone and identification of probe The primers were designed based on the published nucleotide sequences of PRRSV, CSFV, PCV-2, PPV, PRV, JEV and influenza A virus in Genbank. The virus DNA or cDNA was amplified by using high-fidelity DNA polymerase, then ligated with pMD 18-T Simple Vectors to construct recombinant plasmids, respectively. The recombinant plasmids were identified by PCR and sequencing. Aliment reports showed that the sequence had 89.1 to100 percent identities with relevant virus strains, respectively.Part 2: Construction and preliminary application of diagnostic DNA macroarray in porcine viral respiratory diseaseUsed the recombinant plasmids constructed in part 1 to prepare PRRSV, PRV, PCV-2 and SIV probes, and then to prepare diagnostic DNA macroarray for porcine viral respiratory disease.Biotin-11-dUTP concentration options: The target of PRRSV, PRV, PCV-2 and SIV were labeled by Biotin-11-dUTP in PCR. The final concentrations of Biotin-11-dUTP in PCR system were 40μM、50μM、70μM and 100μM, respectively. It was showed that all the products of four PCR systems could get positive hybridization signals. And the signals were increased with the concentration. The result indicated that 70μM concentration (at 35% level of substitution) could meet the requirements of signal analysis.Hybridization temperature options: The target of PRRSV, PRV, PCV-2 and SIV were更多还原