绵羊肺炎支原体免疫层析血清学和病原分子诊断技术的研究

发布时间：2018-05-16 23:43

本文选题：绵羊肺炎支原体 + 胶体金免疫层析技术　；参考：《石河子大学》2017年硕士论文

【摘要】：绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)是一种引起山羊、绵羊、大角羊和小反刍动物传染性胸膜肺炎的主要致病菌,主要症状为咳嗽、喘息、逐渐消瘦、肺间质增生等,病程长达数月至数年,四季均可发病。国内首次从四川边区莱斯特种羊(从新西兰、英国引进)体内分离到Mo病原,并在甘肃、宁夏、内蒙、河北、新疆等多个省份先后报道了Mo感染,Mo引起的胸膜肺炎蔓延日趋严重,给养羊业造成巨大的经济损失。因此,建立一种快速、高敏的检测方法对该病的快速筛查和有效预防具有重要意义。本文从血清学和病原分子生物学两方面入手,探究了以侧向层析为基础的Mo快速检测方法。本研究表达并纯化了绵羊肺炎支原体的infA、Met、MO-1、MO-2四种原核表达蛋白,培养绵羊肺炎支原体后分别提取全菌蛋白和膜蛋白。以所有蛋白分别作为检测抗原组装捕获法胶体金试纸条:胶体金标记金黄色葡萄球菌A蛋白(SPA),检测抗原包被检测线(T线),鸡抗SPA抗体包被质控线(C线)。以infA、Met、MO-1、MO-2蛋白分别作为检测抗原组装双抗原夹心法胶体金试纸条:胶体金标记检测抗原,同时检测抗原包被T线。血清样本加于试纸条上样垫,10~15 min读取结果。试纸条经测试有效后筛选检测抗原,建立Mo的胶体金免疫层析检测方法。结果显示:infA、Met重组蛋白作捕获法的检测抗原时,阳性样品结果明显,阴性样品假阳率较高,约50%。作夹心法的检测抗原时,阳性反应结果较弱,且有假阳性出现;MO-1、MO-2作捕获法的检测抗原时,阳性反应较弱,且有假阳性,与夹心法结果一致;Mo的全菌蛋白和膜蛋白作捕获法的检测抗原时,特异性较好,前者的检出率高于后者。因此,本研究以Mo的全菌蛋白作为检测抗原,初步建立了Mo的捕获法胶体金免疫层析方法,这种方法对其它病原菌感染的血清均显阴性,灵敏度为1:100,表明其特异性和灵敏度较好。没有批内差和批间差,重复性和稳定性良好。本文初步建立了Mo胶体金免疫层析方法,进一步优化后可在基层畜牧兽医站推广使用。本研究结合降落PCR(Touchdown PCR,TD PCR)和侧向层析技术(lateral flow assay,LFA)建立了Mo病原分子生物学检测方法。两条特异性引物5'端分别标记地高辛和生物素,以提取的基因组为模板进行TD PCR;胶体金标记抗地高辛抗体,检测线和质控线分别包被链霉亲和素和羊抗鼠IgG,组装成LFA方法胶体金核酸层析试纸条。将TD PCR产物滴加于核酸试纸条上样垫,在15 min内读取检测结果,通过对各环节的调整优化,建立了检测Mo核酸的TD PCR-LFA快速检测方法。结果显示:建立的TD PCRLFA方法仅对Mo病原呈阳性,其他病原菌均为阴性,表明该方法特异性良好。其检测下限为20.6 ng/ml,敏感性较强。TD PCR批间产物、批间试纸条读取结果一致,重复性和稳定性较好。本研究建立的Mo检测方法整合了TD PCR的特异性、敏感性和金标条快速、简单的特性,在2 h内可完成检测。为建立适用于基层和畜牧兽医站的病原分子学诊断方法奠定了基础。
[Abstract]:Mycoplasma ovipneumoniae (Mo) is a major pathogenic bacteria causing infectious pleuropneumonia in goats, sheep, big horn sheep and small ruminants. The main symptoms are cough, wheezing, gradual emaciation and interstitial lung hyperplasia for several months to several years. It is the first time in Leicester from Sichuan border region. Mo pathogens were isolated from New Zealand and Britain) and Mo infection was reported in many provinces in Gansu, Ningxia, Inner Mongolia, Hebei, Xinjiang and other provinces. The spread of pleuropneumonia caused by Mo was becoming more and more serious, causing huge economic losses to the sheep industry. Therefore, a fast speed, Gao Min detection method for rapid screening and effective prevention of the disease was established. This paper, starting with two aspects of serology and pathogenic molecular biology, explores the rapid detection of Mo based on lateral chromatography. This study expresses and purify the four prokaryotic expression proteins of Mycoplasma sheeppneumoniae, infA, Met, MO-1, MO-2, and extracts whole bacterial protein and membrane protein after the culture of Mycoplasma pneumoniae. All proteins were detected as antigen assembly capture colloid gold strip, colloid gold labeled Staphylococcus aureus A protein (SPA), detection of antigen envelope detection line (T line), chicken anti SPA antibody package on the quality control line (C line). InfA, Met, MO-1, MO-2 protein as antigen assembled double antigen sandwich colloid gold test strip, respectively: colloidal gold mark The antigen was detected and the antigen was detected by T line. The serum samples were added to the sample pad and 10~15 min to read the results. The test paper was tested effectively after testing the antigen and established the colloidal gold immunochromatography detection method of Mo. The results showed that the positive sample results were obvious when infA and Met recombinant protein was used as the capture method to detect the antigen, and the negative samples were false positive. When 50%. was used as a sandwich method to detect antigen, the positive reaction results were weak and there were false positive. When MO-1, MO-2 was used as a capture method to detect antigen, the positive reaction was weak, and there was false positive, which was in accordance with the result of sandwich method. The specificity of the whole bacterial protein and membrane protein of Mo was better than that of the capture method. The detection rate of the former was higher than that of the latter. Therefore, in this study, the total bacterial protein of Mo was used as the antigen to detect the antigen, and a colloidal gold immunochromatography for the capture of Mo was initially established. This method was negative to the serum of other pathogenic bacteria, and the sensitivity was 1:100. It showed that the specificity and sensitivity were good. There was no difference in batch and batch difference, and the reproducibility and stability were good. This paper was preliminary. The Mo colloidal gold immunochromatography (GI) was established, which could be further optimized at the grass-roots animal husbandry and veterinary station. This study combined with the landing PCR (Touchdown PCR, TD PCR) and lateral chromatography (lateral flow assay, LFA) to establish a Mo molecular biological detection method. Two specific primers were labeled with digoxin and biotin, respectively. The genome was taken as a template for TD PCR, and the colloidal gold was labeled with digoxin antibody. The detection line and the quality control line were packed by streptomycin and Sheep anti mouse IgG, respectively, and assembled into a LFA colloid gold nucleic acid test paper. The TD PCR products were added to the sample pad of nucleic acid test paper, and the test results were read in 15 min, and the adjustment of each link was optimized. The rapid detection method of TD PCR-LFA for detecting Mo nucleic acid was established. The results showed that the established TD PCRLFA method was only positive for Mo pathogen and the other pathogenic bacteria were negative, which showed that the method had good specificity. The detection limit of the method was 20.6 ng/ml, the sensitivity of.TD PCR inter batch products was stronger, the reading results of the batch test paper were consistent, repeatability and stability were better. Good. The Mo detection method established in this study integrates the specificity of the TD PCR, the sensitivity and the rapid and simple features of the gold bar, and can be detected within 2 h. It lays the foundation for the establishment of a diagnostic method for the pathogen of grass roots and animal husbandry and veterinary medicine.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.26

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