小反刍兽疫血清抗体免疫过氧化物酶单层试验检测方法的建立

发布时间：2018-06-24 12:51

本文选题：小反刍兽疫 + 免疫过氧化物酶单层试验　；参考：《中国农业科学院》2015年硕士论文

【摘要】：小反刍兽疫(Peste des petits ruminants,PPR)是由副粘病毒科麻疹病毒属的小反刍兽疫病毒(Pest des petits ruminants virus,PPRV)引起的一种急性、高度接触性烈性传染病,主要感染山羊和绵羊等小反刍动物。PPR自1942年在非洲被发现以来,不断向全球蔓延。2007年7月,我国首次在西藏爆发PPR疫情,2013年至2014年全国19个省区再次爆发PPR疫情,对我国养殖业造成巨大经济损失。2015年4月,在科特迪瓦首都阿比让举行的动物卫生专家会议一致同意,尝试在全球消除小反刍兽疫病毒。因此,尽快开展小反刍兽疫的病原学、流行病学、诊断与预防控制技术的综合性研究,建立快速敏感的诊断方法对PPR的防控具有深远的社会和经济意义。目前针对PPRV的诊断方法较多,其中血清学检测方法主要是病毒中和试验(virus neutralization test,VNT)和竞争ELISA方法,但前者存在操作周期长,因操作活病毒而设施要求高等缺点;后者存在价格较贵、制备复杂等缺点。免疫过氧化酶单层试验(immunoperoxidase monolayer assay,IPMA)具有快速、敏感、特异、操作简单的特点,已应用于多种病原的血清临床样本检测。但国内外还没有建立针对PPRV的IPMA检测方法,因此建立PPRV-IPMA检测方法对于PPR疫情的监测和防控具有一定的重要意义。为建立针对PPR的IPMA检测方法,本研究首先建立了稳定表达山羊SLAM的BHK-21细胞系(BHK-gSLAM)。rPPRV/GFP感染该细胞系后,能复制增殖且形成明显的合胞体,而在亲本细胞上无该现象。Western blot试验表明该细胞系能稳定表达SLAM蛋白至少20代。在细胞系建立的基础上,通过感染rPPRV/GFP制备抗原检测板,建立了IPMA检测方法,具体步骤如下:首先比较了Vero细胞和BHK-gSLAM两者在IPMA试验中的差别,结果表明,在IPMA反应板制备过程中,同等剂量的rPPRV/GFP感染Vero和BHK-gSLAM细胞,后者病变形成时间更早,并形成易于观察的合胞体;BHK-gSLAM细胞生长更紧密,形成的CPE更明显,其在AEC染色后更易于观察,因此最终选择该细胞系用于IPMA抗原检测板的制备。其次,本研究优化并确定了IPMA的最佳接毒剂量为104 TCID50/mL,病毒感染72 h后固定制备IPMA反应板,血清最佳起始稀释度为1:10,二抗最佳工作浓度为1:5000。最后,用该IPMA检测方法对198份羊血清(包括临床样本和PPR弱毒疫苗免疫后样本)进行检测,同时以VNT方法作为对照,结果表明,两者的符合率为95.5%。与VNT相比,IPMA的相对敏感性为91%,特异性为100%。综上所述,本研究建立的IPMA方法操作简单,易于观察,检测时间仅为3小时,检测结果与VNT方法符合率较高,同时具有良好的特异性和敏感性,可以替代VNT用于PPRV免疫效果的评价和流行病学调查。
[Abstract]:Peste des petits ruminants (PPR) is an acute and highly contagious disease caused by the measles virus of the genus Pest des petits ruminants virus (PPRV). PPR, which mainly infects small ruminants such as goats and sheep, has been spreading to the world since it was discovered in Africa in 1942. In July 2007, the PPR epidemic occurred for the first time in China in Tibet, and again in 19 provinces and regions in China from 2013 to 2014. In April 2015, an expert meeting on animal health in Abidjan, Ivory Coast, agreed to try to eliminate the small ruminant virus globally. Therefore, it is of great social and economic significance to develop a comprehensive study on the etiology, epidemiology, diagnosis and prevention and control of small ruminant disease as soon as possible, and to establish a rapid and sensitive diagnostic method for the prevention and control of PPR. At present, there are many diagnostic methods for PPRV, of which the serological detection methods are mainly virus neutralization test (virus neutralization testVNT) and competitive Elisa. However, the former has the disadvantages of long operation cycle and high facility requirements due to the operation of live virus. Complex preparation and other shortcomings. The immunoperoxidase monolayer test (immunoperoxidase monolayer assayma) has been applied to the detection of various pathogens in serum because of its rapidity, sensitivity, specificity and simple operation. However, there is no IPMA detection method for PPRV at home and abroad, so it is of great significance to establish PPRV-IPMA detection method for PPR epidemic situation monitoring and prevention and control. In order to establish IPMA detection method for PPR, BHK-21 cell line (BHK-gSLAM), rPPRV / GFP, which expressed goat slam stably, could replicate and proliferate and form distinct syncytial cells after infecting the cell line. Western blot assay showed that the cell line could stably express slam protein for at least 20 passages. Based on the establishment of the cell line, the antigen detection plate was prepared by rPPRV / GFP infection, and the IPMA detection method was established. The specific steps are as follows: firstly, the differences between Vero cells and BHK-gSLAM in IPMA test were compared. The results showed that, during the preparation of IPMA reaction plate, the difference between Vero cells and BHK-gSLAM in IPMA test was compared. The same dose of rPPRV / GFP was infected with Vero and BHK-gSLAM cells, the latter developed earlier, and formed the easily observable syncytial cells BHK-gSLAM cells. The formation of CPE was more obvious, and it was easier to observe after AEC staining. Therefore, the cell line was selected for the preparation of IPMA antigen detection plate. Secondly, the optimal dose of IPMA was 104TCID 50 / mL. The IPMA reaction plate was fixed at 72 h after infection. The optimal initial dilution of IPMA was 1: 10, and the optimal working concentration of the second antibody was 1: 5000. Finally, 198 sheep serum samples (including clinical samples and PPR attenuated vaccine samples) were detected by the IPMA method, and the VNT method was used as control. The results showed that the coincidence rate of the two methods was 95.55g. Compared with VNT, the relative sensitivity of IPMA was 91 and the specificity was 100. In conclusion, the IPMA method established in this study is simple to operate, easy to observe, and the detection time is only 3 hours. The detection results are in good agreement with the VNT method and have good specificity and sensitivity. VNT can be used to evaluate the immune effect of PPRV and to investigate the epidemiology of PPRV.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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