【摘要】：近年來，禽類免疫抑制病在我國養(yǎng)禽業(yè)中迅速蔓延，嚴重地威脅著養(yǎng)禽業(yè)的健康發(fā)展，禽類免疫抑制病在臨床上主要依靠疫苗和藥物來防控和治療；但其中一些疫病迄今為止仍無有效的疫苗和藥物，此類疫病在臨床上主要通過對病原進行檢測，從而達到凈化和防控的目的。本研究基于多重PCR技術，建立了雞傳染性貧血病、禽網狀內皮組織增殖病和區(qū)分禽白血病A、C、D亞群的基因芯片檢測方法。 根據NCBI收錄的雞傳染性貧血病毒、禽網狀內皮增生癥病毒與禽白血病病毒A、C、D亞群參考毒株的序列，設計合成3對特異性擴增引物，將下游引物進行Cy3熒光標記，建立多重PCR體系；并參考目的序列內保守區(qū)域，設計合成10條寡核苷酸探針，按照矩陣設計，點制在醛基化玻璃基片上，制作寡核苷酸探針基因檢測芯片；提取病毒核酸，，進行多重PCR擴增后與探針進行雜交，然后用熒光檢測儀掃描并分析結果。 經雜交反應后，芯片能夠同時檢測雞傳染性貧血病病毒和禽網狀內皮增殖病病毒，并能區(qū)分禽白血病病毒A、C、D亞群，其靈敏度能夠到達107copies/mL，并且具有較高的特異性�；蛐酒瑢�450份臨床樣品檢測的結果與ELISA抗體檢測結果相比符合率高。本研究結果證明，基因芯片技術是一種有效地檢測雞傳染性貧血病毒和禽網狀內皮增殖病病毒，并能區(qū)分禽白血病病毒A、C、D亞群的方法，為今后在臨床應用中快速鑒別診斷雞傳染性貧血病病毒、禽網狀內皮增生癥病病毒與禽白血病病毒A、C、D亞群提供可行性。 通過本研究方法建立了禽白血病基因芯片，對同一只雞的全血、血清、蛋清和泄殖腔棉拭子等進行跟蹤檢測。檢測結果顯示，基因芯片方法檢測全血的陽性率為69.23%；ELISA試劑盒檢測蛋清樣品中p27抗原的陽性率為23.08%。芯片檢測率顯著高于ELISA試劑盒檢測率，且全血是用于禽白血病基因芯片檢測的最優(yōu)材料。
[Abstract]:In recent years, avian immunosuppressive diseases have spread rapidly in poultry industry in our country, which seriously threaten the healthy development of poultry industry. Poultry immunosuppressive diseases mainly rely on vaccines and drugs to prevent and treat them in clinic. However, there are no effective vaccines and drugs for some of the epidemic diseases so far, and this kind of disease can be purified and controlled by the detection of the pathogen in clinical practice, so as to achieve the purpose of purification and prevention and control. In this study, a gene chip method was developed for detection of avian infectious anemia, reticuloendotheliosis and differentiated avian leukemia A, C, D based on multiplex PCR. According to the sequences of avian infectious anemia virus, avian reticuloendotheliosis virus and avian leukemia virus A, C, D included in NCBI, three pairs of specific amplified primers were designed and synthesized, and the downstream primers were labeled with Cy3 fluorescence. Establish multiple PCR system; Ten oligonucleotide probes were designed and synthesized according to the conserved region of the target sequence. According to the matrix design, the oligonucleotide probes were fabricated on the aldehyded glass substrate and the oligonucleotide probe gene detection chip was fabricated. The viral nucleic acid was extracted and hybridized with the probe by multiplex PCR. Then the results were scanned and analyzed by fluorescence detector. After hybridization, the microarray can detect avian infectious anemia virus and reticuloendotheliosis virus simultaneously, and can distinguish avian leukemia virus A, C, D subpopulations. The sensitivity of the microarray can reach 107 copies / mL and has high specificity. Compared with the results of ELISA antibody detection, the coincident rate of the results detected by gene chip was higher than that of the other 450 clinical samples. The results show that gene chip technique is an effective method to detect avian infectious anemia virus and reticuloendotheliosis virus, and can distinguish avian leukemia virus A, C, D subsets. To provide feasibility for the rapid differential diagnosis of avian infectious anaemia virus, reticuloendothelial hyperplasia virus and avian leukemia virus A, C, D in clinical application in the future. The whole blood, serum, egg white and cloacal cotton swabs of the same chicken were tracked and detected by using the gene chip of avian leukemia. The results showed that the positive rate of the whole blood detected by gene chip was 69.23% and the positive rate of p27 antigen in egg white samples was 23.08% by Elisa kit. The detection rate of microarray was significantly higher than that of ELISA kit, and the whole blood was the best material for detection of avian leukemia gene chip.
【學位授予單位】：河南科技學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.31

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