【作者】 王小强；

【导师】 李铮； 陈超；

【作者基本信息】 西北大学 ， 微生物学， 2007， 硕士

【摘要】 高发性呼吸系统、生殖系统、神经及免疫系统主要病毒性病原的混合感染和持续感染，使得集约化养猪业疫病流行状况呈现高度复杂化、多样化趋势，进而增加了临床和实验室诊断难度。目前，对猪疫病病毒性病原的诊断仍采用传统的病原分离和免疫血清学方法，其操作费时费力且灵敏度不高。分子生物学诊断方法如：PCR、标记探针杂交等技术的应用，虽然具有灵敏度高、特异性强、快速、简便的特点，但其假阳性率较高且一次仅能检测一种或同时检测两三种病毒。基于上述技术的缺点，本研究应用寡核苷酸芯片技术，结合不对称PCR和间接荧光标记技术，制备一款低密度猪疫病病原诊断基因芯片，以满足目前集约化养猪生产中流行性病疫原快速、准确、高通量的检疫要求。本研究确定集约化养猪业常见的4种猪疫病病毒：猪流感病毒、猪伪狂犬病毒、口蹄疫病毒、猪蓝耳病毒作为检测对象。根据四种不同病毒的保守区域设计扩增短片断(100-300 bp)PCR产物的特异性引物。依据每种短片断PCR产物序列，筛选出四条高度特异、长度相同、Tm值和G+C％含量接近的检测探针，并将其固定在氨基化修饰的片基上，制备成低密度60-mer寡核苷酸诊断芯片。通过标记样本与检测探针在固液相体系中杂交，一次试验可完成对这四种猪疫病病毒的诊断。在实验过程中，对芯片上探针点样浓度的选择，每种病毒选择正义链或是反义链探针作为检测探针以及杂交条件等实验过程进行探索和优化。以上述四种猪疫病病毒为检测模型，对经过优化的猪疫病病毒诊断基因芯片集成系统的灵敏度和特异性进行评估和鉴定。通过一系列试验，本研究取得如下的成果：(1)制作出背景低、固定效率高的(?)病毒性疫病原多重检测的基因芯片；(2)通过优化实验，确定了芯片上探针(?)浓度的最佳浓度为25μM；(3)不对称PCR体系中通过变换上下游引物作为限制性引物，杂交后结果进行比较，最终确定猪流感病毒、猪伪狂犬病毒及口蹄疫病毒以反义链探针作为检测探针，而猪蓝耳病毒则以正义链探针作为检测探针；(4)通过用间接荧光标记技术引入荧光基团，在杂交温度为60℃，杂交时间为10h的条件下，能够获得特异性强、稳定性高的荧光信号；(6)从杂交结果可以看到四种猪疫病病毒均可在在对应的检测探针位置处被检出，没有假阳性结果出现，各个病毒之间也没有出现交叉杂交的现象。芯片检测四种猪疫病病毒靶基因的最低检测限分别是：猪流感病毒7.59×10~2个／mL，猪伪狂犬病毒2.36×10~4个／mL，口蹄疫病毒6个／mL，猪繁殖与呼吸综合症病毒5.05×10~2个／mL。更多还原

【Abstract】 The important porcine pathogens are frequently complicated with an increased incidence of viral superinfections, which endanger respiratory system, neural system, and reproductive system in the intensive swine industry. Furthermore, the difficulty of laboratory and clinical diagnosis of porcine pathogenic infections would be increased. The rapid and exact diagnosis of epidemic disease and adopting relevant measures is a precontion for loimic prevention. Current routine techniques for identification and detection of porcine pathogenic viruses are still based on virus isolation in tissue culture and serological method, but they are laborious, time-consuming and have a relatively low sensitivity. The advent of several molecular methods are established as a specific, fast and sensitive method for virus detection, such as PCR and southern blotting, but the incidence of false positivity is high and the capacity of these assays are limited. To address some of the limitations of existing methods for pathogens detection, in this paper, a low-density oligonucleotide microarray was develpoed coulped with asymmetric PCR and indirect labeled method for the rapid, accurate and high-throughput detection of epidemic viral pathogens in the swine industry.In this study, four familiar swine virues were chosen as the investigative targets, including swine influenza virus (SIV), porcine pseudorabies virus (PRV), foot-and-mouth disease virus (FMDV) and porcine reproductive and respiratory syndrome virus (PRRSV). The specific primers were designed to amplify 100-300 bp lengh fragments based on conservative regions of each swine virus. The oligonucleotide probes immobilized on the matrix were designed based on target symmetric PCR products, at the same time ensured the same length (60-mer) as well as approximately identical GC content and Tm for each capture probe. The oligonucleotide probes immobilized on the matrix were designed using DNAstar and Oligo 6.0 software based on target symmetric PCR products, at the same time ensured the same length (60-mer) as well as approximately identical GC content and Tm for each capture probe. After the labelled samples were subjected to solid and solution phase hybridization, four swine virues could be diagnosed in one assay. In this paper we also explored optimization of the following experiment processes: spotting concentration on the microarrays, scale of of restrictive and unrestrictive primers in asymmetric PCR mixture, choice of sense stand or anti-sense probe as capture probe and hybridization condition. Finally, the sensitivity and specificity of oligonucleotides microarray were appraised using this four kinds of pathogens as the detected model.This research attains the following achievements: (1) The diagnostic oligonucleotide microanay has the virtues of low background and high probed binding efficiency; (2) Through the optimized experiments, the optimal spotting concentration is confirmed to be 25μM; (3) The hybridization results revealed when the reverse primer was ascertained to be restrictive for PRV, SIV, FMDV, while the forward primer was ascertained to be restrictive for PRRSV, at the same time with a restrictive /unrestrictive primer ratio of 1: 100, the specificity of hybridization signals was the most strongest. (4) The hybridization temperature was at 60℃, the hybridization time was for 10h, the strong and stable fluorescent signal could be gained; (5) The hybridization results showed this technique could identify and distinguish the four swine viruses, there were no false positive results and cross hybridization. The detection limit of the oligonucleotide microarray system were 1.63×10~3 gene copy numbers for SIV, 7.08×10~4 gene copy numbers for PRV, 18 gene copy numbers for FMDV, 1.52×10~3 gene copy numbers for PRRSV.更多还原