【作者】 侯义宏；

【导师】 钱永华； 韩雪清；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2007， 硕士

【摘要】 禽流感(Avian influenza,AI)是由流感病毒属的禽流感病毒(Avian influenza virus,AIV)引起的禽类感染和疾病的总称,尤其是高致病性禽流感(High pathogenic avian influenza, HPAI)可导致高达100%的发病率和死亡率,并可感染人和致人死亡,不仅给世界各国的畜牧业和国际贸易带来巨大的经济损失,而且严重威胁着人类健康。而AIV血清型众多,抗原变异性强,宿主范围广,各亚型间几乎没有交叉保护性,是进行诊断和防控的难点。基因芯片技术作为一种比较系统、完善的检测技术成为近年来研究的热点,为建立快速、敏感、特异的AIV所有亚型分型检测技术,及早发现新流行亚型,有效监控禽流感,本论文对AIV分型基因芯片技术进行初步研究。论文由两个部分构成,第一部分是设计25对引物,对AIV多重RT-PCR进行研究;第二部分是进行多重不对称RT-PCR扩增,设计探针制备病毒分型基因芯片,对AIV 25个亚型分型基因芯片检测方法进行初步研究。主要实验和结果如下:(1)根据Genebank中已发表的AIV不同亚型的基因保守序列,设计和合成AIV 25对特异性引物;对已获得标准毒株的亚型,提取病毒RNA进行多重RT-PCR扩增;对未能获得标准毒株的亚型,设计和合成用于搭桥PCR技术的203条寡核苷酸片断,采用搭桥PCR技术获得各亚型目的基因,将其随机分成3组进行多重RT-PCR扩增;进一步特异性和敏感性试验,对AIV多重RT-PCR扩增进行研究;用标准毒株的病毒RNA(不包括H16和N7亚型)对多重RT-PCR进行验证试验,应用此多重RT-PCR和病毒分离经典方法对395份(4省20多个地区)口岸检样进行平行检测。结果表明:本试验通过搭桥PCR技术成功获得了未能获得AIV标准毒株17个亚型的目的基因,与禽类常见6种病毒(IBV﹑ARV﹑IBDV、NDV、FPV、MDV)扩增均为阴性,其H5N1的敏感度可达247 pfu,AIV参考毒株H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H12、H14、H15、N1、N2、N3、N4、N6、N8、N9亚型均扩增出较亮的目的条带,H11、H13和N5亚型目的条带稍弱,平行检测结果完全吻合。(2)在多重RT-PCR基础上对通用引物介导的多重不对称RT-PCR进行研究;基于扩增的靶序列和Genebank中已发表AIV不同亚型的基因序列,设计AIV分型探针和质控探针,对其修饰并合成,用芯片点样仪将探针固定到玻片上,制备AIV基因检测芯片;并测定其特异性和敏感性,对AIV分型基因芯片检测方法进行初步研究。结果显示:与IBV﹑ARV﹑IBDV、NDV、FPV、MDV均无交叉反应,AIV各亚型探针位点均出现各自的检测信号,扩增目的片段敏感性可达2.5 ng;该方法具有较好的特异性和敏感性。更多还原

【Abstract】 All the diseases and infection caused by avian influenza virus(AIV) are called avian influenza.Especially high pathogenic avian influenza(HPAI) can infect and cause disease in humans with a fatal illness.Not only HPAI bring a big economical loss to international trade, but also it threat badly human.AIV included twenty-five subtypes, with no cross-protection each other. In order to develop rapid, sensitive, specific identification and subtypeing, DNA microarray for AIV were studied on this paper.The paper has two parts, one is the study on the multiplex reverse transcriptase- PCR(RT-PCR) of AIV. The other is the primary study on the subtying of AIV using DNA microarray method. The main experiments and results are as follows:(1)Based on the conserved region sequences of the AIV, twenty-five pairs of primers were carefully designed. The primers were synthesized. Viral RNA of H1, H3, H5, H6, H7, H9, N1and N2 were extracted and performed multiplex RT-PCR amplication. The target genes of other subtypes AIV were obtained by overlapping PCR. The results showed that target gene of 17 AIV subtypes were achieved successfully. Then they were random divided into three groups and develop multiplex RT-PCR respectively. Reference virual RNA of AIV run multiplex RT-PCR amplication except H16, N7 subtypes, H11, H13 and N6 subtypes cDNA bands were dim, the other were brighten bands. The sensitivity and specificity of the method were evaluated by using NDV, IBV, ARV, IBDV, MDV, FPV, the results showed that the method was very specific and sensitive. the sensitivity of H5N1 is up to 247 PFU. The method was also used to detect 395 avian field samples from more than 20 regions of 4 different provinces, and the detection results was consistent with the conventional virus isolation method.(2)Multiplex asymmetric RT-PCR were performed using universal primers, and special primers. Based on the conserved region sequences of the AIV, subtyping probes and quality control probes were designed and synthesized. After spotting on the glassslide, the microarray has been developed by application of several control and gene-specific probes of AIV. The specificity of the gene microarray were evaluated by using NDV, IBV, ARV, IBDV, MDV, FPV. Gene-specific probes of AIV indicates good fluorescence signal in their location, with no cross-reaction to other subtype or clinical relvant virus. the results showed that the method was very specific.The sensitivity of the cDNAs is 2.5 ng.更多还原