【作者】 杨林；

【导师】 赵德明；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2005， 博士

【摘要】 猪瘟(CSF)是由猪瘟病毒(CSFV)引起的一种猪病毒性传染病,猪瘟兔化弱毒疫苗有效地控制了猪瘟在我国的急性发生和大流行。但自上世纪80年代以来,我国开始出现了温和、非典型性猪瘟。究其原因,可能与CSFV出现了新的变异株等因素有关。尤其是亚临床感染猪,依靠常规诊断方法很难确诊并剔除此类病猪,给CSF的防制工作带来新的困难。因此,有必要建立一种新的迅速、准确诊断CSF的实验室诊断系统,为在我国彻底消灭CSF奠定基础。 基因芯片(Gene Chip)是生物技术与电子芯片融合的结晶,是由固定于固相载体(如硅片、玻璃、塑料等)表面的核苷酸阵列构成的一种新型微型器件。由于其快速、微量、准确的应用优点,正在成为新一代的自动化医学检验工具。通过设计针对基因的探针,与被检测基因的碱基序列互补匹配,可快速、准确地检测到基因,用于疫病的诊断。 本研究采用以基因测序为基础的系统进化树分析方法,对黑龙江省部分地区猪瘟病毒流行株进行分离鉴定和E2基因序列分析,并与传统的石门强毒和猪瘟兔化弱毒在抗原基因上存在的变异进行比较。研究结果表明:病料为CSFV感染,CSF流行毒株和疫苗株在核苷酸和氨基酸序列上存在一定的差异,E2基因部分序列接近Alfort,与Brescia同源性最低。该试验从核酸和蛋白质水平上研究了黑龙江省部分地区猪瘟病毒流行株与其它标准毒株之间的同源性及其变异情况,揭示了在该省猪瘟流行的本质,为实施科学有效的猪瘟防制措施提供依据。 本研究通过RT-PCR扩增CSAV相当保守的NS基因作靶DNA制作芯片,将被检样品在PCR过程中用Cy-5标记,得到DNA产物与芯片杂交,达到检测CSFV的目的,建立CSF基因芯片诊断方法;同时通过研究不同靶探针浓度对检测结果的影响,确立最佳反应条件靶探针浓度为1 μg/μl;分析芯片诊断技术的特异性和敏感性等技术指标:与RT-PCR方法和间接荧光抗体试验方法进行比较,并开展了初步应用。本研究建立的CSFV基因芯片诊断技术,可以成功地检测到CSFV保守区基因,荧光信号清晰,敏感性利特异性高,实际应用效果奸,是一种检出率高的诊断方法。 总之,本研究将基因芯片技术应用于动物疫病的诊断,成功建立了CSFV基因芯片诊断技术平台,解决了CSF诊断中的难题,具有重大的理论意义和应用价值。为今后利用基因芯片开展动物疫病诊断和研究提供理论依据,也为控制和消灭CSF奠定基础。但要实现基因芯片在动物疫病方面的广泛应用,还要做更深入的研究。更多还原

【Abstract】 CSF(Classical swine fever), the virulent infectious disease caused by CSFV (Classical swine fever virus), had been controlled effectively by CSFV strain vaccine in the country. But atypical HC and inapparent CSF began to appear after 1980s, which had resulted in great loss. This result may have something to do with the appearance of the new variation virus of CSFV and other factors. Even sub-clinic infections swine is difficult to diagnose and eliminate them, depending on the general diagnosis method and bring new problem to the prevention and treatment. Thus it is necessary to establish a new, swift and reliable diagnosis system in the lab to pave a way for completely annihilating CSF.Gene chip or microarray, the combination of biology and microelectronics technology, is a kind of new micro-device. It is composed of DNA arrays immobilized on solid supports. Because of its many virtues, such as rapid, sensitive and accurate, gene chip plays an important role in the study of diseases. Through the match of mutation probes and sequences, we can detect the mutation quickly, lightly and accurately. The detection can effectively instruct disease diagnosis.The study utilized phylogenetic tree based on gene sequence to isolate and identify CSFVs in some areas of Heilongjiang province and sequence E2 gene of every virus. The results showed in the following: Doubtful materials were infected with CSFVs, the sequences of E2 genes nearer to Alfort, the homology with Brescia was the lowest. We researched the homology and variation among prevalent CSFVs on nucleic acid and protein level in some areas in the province. At the same time, the antigenic variation between traditional Shimen virus and virus cultivated in rabbits was compared in order to disclose the prevalent essence of CSF in the province and tried to acquire new efficient methods for preventing and treating CSF better and better.In this study a microarray-based assay for the detection CSFV was developed. The captor-DNA fragments, which were reamplified from CSFV C stain, were spotted on a glass-bound microarray. Cy5-labeled fluorescent PCR production were hybridized to these captor-DNA on microarray. At the same time, we studied the effects of the different concentration of the captor-DNA on the detecting result, confirmed the concentration of captor-DNA is 1μg/μl, established the option reaction condition, analyzed the specialty and sensitivity and compared it with RT-PCR and IFA experiment method. The result show that microarray diagnosis method of CSFV established in this research can successfullyverify the conservative sequence of the CSFV. The signal of the fluorescence is clear, the specialty is high.In fact it is an efficient diagnosis method.To sum up, this study successfully established a diagnosis technique platform of microarray and solved the difficult problem for CSFV, with afford a theoretical base for prevention and diagnosis of animal’s epidemic diseases, control and annihilate CSF.But in order to abroad applicate microarray in animal disease,we need to do more reseach.更多还原