鸡三种免疫抑制病基因芯片检测方法的建立
发布时间：2019-03-26 10:00
【摘要】：近年来，禽类免疫抑制病在我国养禽业中迅速蔓延，严重地威胁着养禽业的健康发展，禽类免疫抑制病在临床上主要依靠疫苗和药物来防控和治疗；但其中一些疫病迄今为止仍无有效的疫苗和药物，此类疫病在临床上主要通过对病原进行检测，从而达到净化和防控的目的。本研究基于多重PCR技术，建立了鸡传染性贫血病、禽网状内皮组织增殖病和区分禽白血病A、C、D亚群的基因芯片检测方法。 根据NCBI收录的鸡传染性贫血病毒、禽网状内皮增生症病毒与禽白血病病毒A、C、D亚群参考毒株的序列，设计合成3对特异性扩增引物，将下游引物进行Cy3荧光标记，建立多重PCR体系；并参考目的序列内保守区域，设计合成10条寡核苷酸探针，按照矩阵设计，点制在醛基化玻璃基片上，制作寡核苷酸探针基因检测芯片；提取病毒核酸，，进行多重PCR扩增后与探针进行杂交，然后用荧光检测仪扫描并分析结果。 经杂交反应后，芯片能够同时检测鸡传染性贫血病病毒和禽网状内皮增殖病病毒，并能区分禽白血病病毒A、C、D亚群，其灵敏度能够到达107copies/mL，并且具有较高的特异性。基因芯片对450份临床样品检测的结果与ELISA抗体检测结果相比符合率高。本研究结果证明，基因芯片技术是一种有效地检测鸡传染性贫血病毒和禽网状内皮增殖病病毒，并能区分禽白血病病毒A、C、D亚群的方法，为今后在临床应用中快速鉴别诊断鸡传染性贫血病病毒、禽网状内皮增生症病病毒与禽白血病病毒A、C、D亚群提供可行性。 通过本研究方法建立了禽白血病基因芯片，对同一只鸡的全血、血清、蛋清和泄殖腔棉拭子等进行跟踪检测。检测结果显示，基因芯片方法检测全血的阳性率为69.23%；ELISA试剂盒检测蛋清样品中p27抗原的阳性率为23.08%。芯片检测率显著高于ELISA试剂盒检测率，且全血是用于禽白血病基因芯片检测的最优材料。
[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.
【学位授予单位】：河南科技学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31
本文编号：2447434
[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.
【学位授予单位】：河南科技学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31
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