东部、西部马脑脊髓炎病毒抗体及核酸快速检测技术

发布时间：2018-05-10 06:19

  本文选题：东部、西部马脑脊髓炎病毒 + 抗体　； 参考：《中国农业大学》2015年博士论文

【摘要】：东部、西部马脑脊髓炎病毒可引起人和动物的严重感染,是我国出入境检验检疫中的一类检疫对象,我国目前尚未有这两种病的病例报道。随着马匹国际贸易和国际赛事的日益繁荣,来自不同的国家和地区、健康状况不同的马匹可能进口到我国,因此传入风险不断加大。对此,除实施严格的隔离检疫制度之外,还需研制快速、准确的检测技术作为储备。本研究针对口岸检疫实际需求,研究建立了东部、西部马脑脊髓炎病毒荧光RT-PCR检测方法和IgM抗体间接ELISA检测方法并应用于口岸临床样品的检测。采用TaqMan方法,在对东部、西部马脑脊髓炎病毒基因组序列多重比对基础上,设计合成简并引物和探针。通过优化各项反应条件,建立了两种分别针对东部、西部马脑脊髓炎病毒的实时荧光RT-PCR检测方法。为评价方法的灵敏度,通过体外转录制备了东部、西部马脑脊髓炎病毒保守区序列的核酸标准品cRNA,经稀释、分装、定量、均匀性和稳定性检验后,用于方法的灵敏度评价。结果显示建立的两种方法灵敏度均达到10拷贝/反应。特异性试验结果显示建立的方法特异,与收集的相关马病病原核酸无交叉反应。对临床样品的检测中进一步证实研制的荧光RT-PCR检测方法快速、敏感、特异且重复性好,检测时限3个小时以内。经基因扩增分别获取了东部、西部马脑脊髓炎病毒E2蛋白编码基因,进而将获得的E2基因分别克隆到原核表达载体,然后转化E.coli Rosseta细胞后进行诱导,SDS-PAGE结果显示外源基因获得表达。将表达产物作Western-blot鉴定,结果表明表达产物能够分别与东部、西部马脑脊髓炎病毒IgM阳性血清产生反应,表明其具有反应原性。以纯化后的重组蛋白分别包被酶标板,使用东部、西部马脑脊髓炎病毒IgM阴、阳性血清建立了检测东部马脑脊髓炎、西部马脑脊髓炎IgM抗体的间接ELISA方法。对建立的方法的敏感性、特异性进行评价后,经板内重复试验和板间重复试验证明检测方法重复性良好,并使用研制的两种ELISA检测方法对已知阴、阳性马血清样品进行了验证检测,检测结果与预期完全一致。
[Abstract]:Equine encephalomyelitis virus (EMDV) in the east and west of China can cause serious infection in human and animal. It is a kind of quarantine object in the entry and exit inspection and quarantine of our country. At present, there are no cases of these two diseases reported in our country. With the increasing prosperity of international trade and race of horses, horses with different health status may be imported into China from different countries and regions, so the risk of import is increasing. Therefore, in addition to strict quarantine and quarantine system, rapid and accurate detection technology should be developed as reserve. In order to meet the actual requirement of quarantine at the port, this study established the method of detecting equine encephalomyelitis virus fluorescence RT-PCR in the east and west of China and the indirect ELISA detection method of IgM antibody, and applied it to the detection of clinical samples at the port. TaqMan method was used to design and synthesize degenerate primers and probes on the basis of multiple alignment of equine encephalomyelitis virus genome sequences in the east and west of China. By optimizing the reaction conditions, two real-time fluorescence RT-PCR detection methods for equine encephalomyelitis virus in eastern and western regions were established. In order to evaluate the sensitivity of the method, the standard nucleic acid (cRNAs) of the conserved region of equine encephalomyelitis virus in the east and west of China were prepared by in vitro transcription. After dilution, loading, quantification, homogeneity and stability tests, cRNAs were used to evaluate the sensitivity of the method. The results showed that the sensitivity of the two methods was 10 copies / reaction. The results of specificity test showed that the method was specific and had no cross-reaction with the collected nucleic acid of horse disease. It was further confirmed that the developed fluorescent RT-PCR method was rapid, sensitive, specific and reproducible, and the detection time was less than 3 hours. The E2 protein encoding gene of equine encephalomyelitis virus (EMDV) was obtained by gene amplification, and then cloned into prokaryotic expression vector. Then the E.coli Rosseta cells were transformed and induced by SDS-PAGE. The expression product was identified by Western-blot. The results showed that the expressed product could react with the IgM positive serum of equine encephalomyelitis virus in the east and west of China respectively, which indicated that the product was reactive. The purified recombinant protein was coated with enzyme-labeled plates, and an indirect ELISA method was established for the detection of IgM antibodies against equine encephalomyelitis and equine encephalomyelitis in eastern and western equine encephalomyelitis by using the positive serum of equine encephalomyelitis virus (IgM) in the east and west of the region. The sensitivity and specificity of the established method were evaluated. The results of intraplate repeated test and inter-plate repeated test proved that the method was reproducible, and two ELISA detection methods were used to detect the known negative. Positive horse serum samples were verified and tested, and the results were in accordance with expectations.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S858.21
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本文编号：1868168