【作者】 陈凤梅；

【导师】 牛钟相； 吴时友； 尹燕博；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 本研究用分子克隆方法获得新城疫病毒、禽流感病毒、传染性支气管炎病毒、传染性喉气管炎病毒与鸡毒支原体各一段高度保守基因片段作为探针,用芯片点样仪点样到硝酸纤维膜上,制备成检测基因芯片:提取样品中的RNA进行反转录和生物素标记后滴加到芯片上进行杂交反应,对杂交结果进行扫描分析,可同时对上述五种常见鸡呼吸道疾病做出诊断。 本研究共分为以下三部分: 第一部分 鸡常见呼吸道病诊断基因芯片方法的建立 本部分包括基因芯片的设计与制备、靶基因的标记、芯片杂交时间的选择和芯片的效能评价。通过PCR或RT-PCR方法,分别扩增五种病原的特异性核酸片断,并分别与pMD18—T载体连接,连接产物转化大肠杆菌后,通过PCR法和测序法进行了阳性克隆筛选鉴定。凝胶电泳结果表明,阳性克隆在对应位置可看到特异条带;应用BLAST对扩增产物测序结果与被检病毒核酸序列的进行同源比对,证明与各种被检病毒的大部分毒株同源性在95%以上。提取阳性质粒核酸,扩增纯化后作为探针点在硝酸纤维素膜上,80℃烤膜2h,便完成了基因芯片的制备。在芯片的制备过程中,对不同厂家和不同孔径的膜进行了筛选,结果表明0.65μm的北化膜遇溶液不变形,点不易扩散,效果好于浙江黄岩的膜,本底好于美国进口膜。 针对靶基因的标记尝试了三种核酸标记方法（掺入法、光敏生物素标记法和末端标记法）,试验结果表明用biotin—16—dUTP掺入法进行标记,灵敏度好于光敏生物素标记,特异性好于末端标记。在42℃下对杂交的时间进行了选择,试验表明,最优的杂交时间为1h。 通过对10份阴性样品和20份阳性样品的检测结果表明:阳性参照有明显的杂交信号,阴性参照和空白对照无信号检出,对应位点有明显的杂交信号,芯片的监控系统和疾病诊断系统正常。把NDV培养液作10倍倍比稀释后进行灵敏度检测,可检测到的阳性杂交信号的最大稀释度为10^-10,而凝胶电泳仅能检测到10^-6,这说明芯片的灵敏度是PCR的10⁴倍。分别提取5种病原的核酸进行了特异性试验,用混合引物,单一模板经过RT—PCR和生物素标记后分别与芯片进行杂交,结果表明仅在相应更多还原

【Abstract】 A macroarray technique was developed by preparing highly conserved DNA fragments for the five poultry respiratory tract diseases using molecular cloning methods, and the DNA fragments were spotted onto NC filter to form macroarrays. RNA extracted from samples was reverse-transcripted then followed by PCR amplification and labled with biotin-16-dUTP. The labeled DNA and prepared DNA macroarray were subjected to specific hybridization and the hybridization results were scanned and analyzed with a scanner .The results shows that the macroarray technique could identify and distinguish the five poultry respiratory tract diseases.This research was divided into the following three parts:Part Ⅰ, Establishment of the macroarray techniques in identifying and distinguishing the five poultry respiratory tract diseases. Thedesigning and preparation, labling of target gene, hybridization and scanning of Macroarray, the efficiency appraise of Macroarray were included in this part.We amplified the special sequence and linked them into pMD-18T vector separately,then transformed them into Ecoli and identified them by PCR and detecting sequence.The result of gel electrophoresis shows special strips of positive clone at corresponding site. We compared the sequence with that in the Genbank of NCBI, the result shows that all kinds of detected virus are above 95% in homology .We distilled nucleic acid from positive plasmid and spotted them onto NC filter after purified, then we baked it for 2 hours at 80℃, finally we finished the preparation of Macroarray. We choosed different aperture NC filter from dissimilitude factory during the preparation. As a result, 0.65μm NC filter of ZheJiangHangyan was the best which couldn’t distort and diffuse. The backgrounds was good than that of America. We attempted three kinds of labling methods in the course of detecting samples, such as mixing method, photosensitive biotin method and end labling method. And the result shows that biotin-16-dUTP mixing in sensitivity and specialty is better than photosensitive biotin and end labling method.At the same time, we optimized the hybridization time .The best time is 1hour at 42℃We can conclude that the supervising system and disease diagnosissystem is constant by the detection of 10 shares of negative samples and 20 shares of positive samples. There were evident hybridization signal at positive control site and corresponding site, while no signal at negative site.We diluted the NDV by 10 times aimed at the sensitivity detection. And we could detect hybridization signal at 10’10 times diluting, but the gel can only detect 10"6 time diluting. The result shows that the sensitivity of DNA chip is higher than PCR by 104 times.Nucleic acid were distilled from five kinds of pathogens separately, using reverse transcript PCR amplified them and labled by biotin, in mixing primer and single template .We can only find hybridization signal at corresponding site and no no-specialty hybridization site. Repeating test shows that, every site is good.Part II, Comparison among Macroarry technique, separation and culture, PCR method. We detected 10 positive samples of every kinds pathogen. The positive samples are identified by Oland biological engineer academy. The result shows that separation and culture method is most different from PCR and Macroarry technique (p<0.01), while the radio of the anastomose is above 90% in PCR and Macroarry technique, but the separation and culture is a bit low compared with themPart IE, tentative application of Macroarry technique. We diagnosis the doubtful samples with Macroarry technique and PCR method in Qingdao,Jining,Zhucheng et al.The result shows that, the radio of the anastomose is almost above 90%.At the same time, we compared 60 shares of swab samples and homogenate samples by Macroarry technique. As a result, there was the radio of the anastomose is above 80%.更多还原