【摘要】：近年來(lái)，禽類免疫抑制病在我國(guó)養(yǎng)禽業(yè)中迅速蔓延，嚴(yán)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(lái)防控和治療；但其中一些疫病迄今為止仍無(wú)有效的疫苗和藥物，此類疫病在臨床上主要通過(guò)對(duì)病原進(jìn)行檢測(cè)，從而達(dá)到凈化和防控的目的。本研究基于多重PCR技術(shù)，建立了雞傳染性貧血病、禽網(wǎng)狀內(nèi)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(cè)方法。 根據(jù)NCBI收錄的雞傳染性貧血病毒、禽網(wǎng)狀內(nèi)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(shè)計(jì)合成3對(duì)特異性擴(kuò)增引物，將下游引物進(jìn)行Cy3熒光標(biāo)記，建立多重PCR體系；并參考目的序列內(nèi)保守區(qū)域，設(shè)計(jì)合成10條寡核苷酸探針，按照矩陣設(shè)計(jì)，點(diǎn)制在醛基化玻璃基片上，制作寡核苷酸探針基因檢測(cè)芯片；提取病毒核酸，，進(jìn)行多重PCR擴(kuò)增后與探針進(jìn)行雜交，然后用熒光檢測(cè)儀掃描并分析結(jié)果。 經(jīng)雜交反應(yīng)后，芯片能夠同時(shí)檢測(cè)雞傳染性貧血病病毒和禽網(wǎng)狀內(nèi)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(dá)107copies/mL，并且具有較高的特異性。基因芯片對(duì)450份臨床樣品檢測(cè)的結(jié)果與ELISA抗體檢測(cè)結(jié)果相比符合率高。本研究結(jié)果證明，基因芯片技術(shù)是一種有效地檢測(cè)雞傳染性貧血病毒和禽網(wǎng)狀內(nèi)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(yīng)用中快速鑒別診斷雞傳染性貧血病病毒、禽網(wǎng)狀內(nèi)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(guò)本研究方法建立了禽白血病基因芯片，對(duì)同一只雞的全血、血清、蛋清和泄殖腔棉拭子等進(jìn)行跟蹤檢測(cè)。檢測(cè)結(jié)果顯示，基因芯片方法檢測(cè)全血的陽(yáng)性率為69.23%；ELISA試劑盒檢測(cè)蛋清樣品中p27抗原的陽(yáng)性率為23.08%。芯片檢測(cè)率顯著高于ELISA試劑盒檢測(cè)率，且全血是用于禽白血病基因芯片檢測(cè)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(xué)位授予單位】：河南科技學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.31

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