莱姆病螺旋体重组特异性抗原和RPA检测技术的研究

发布时间：2018-06-10 21:24

本文选题：莱姆病 + 伯氏疏螺旋体　；参考：《中国疾病预防控制中心》2016年硕士论文

【摘要】：莱姆病是由伯氏疏螺旋体引起的蜱传人兽共患病。莱姆病的诊断主要依靠临床症状和实验室辅助诊断。在临床症状不显著的情况下,实验室诊断尤为重要。但是目前国内市场上暂未出现适合于国内莱姆病标准商品化的血清学诊断试剂盒,另外在莱姆病的快速检测方面仍需要提高。本研究的目的主要在于重组表达莱姆病螺旋体特异性抗原并对其进行了血清学诊断评价,通过统计学方法筛选出最合适的有诊断意义的蛋白抗原,为日后研制中国莱姆病免疫学检测试剂盒提供依据。另外建立一种新的基于重组酶聚合酶扩增技术的莱姆病螺旋体核酸快速检测方法,结合侧流层析技术,更简单方便,更有利于莱姆病的快速诊断。我们共选取了5种蛋白抗原,Fla B.g (Fla中央区蛋白),OspC (B.g型和B.a型),P39 B.g, P83-E4 B.g和VlsE B.a,对其进行了克隆表达和纯化。利用免疫印迹实验对重组蛋白进行初步免疫原性检测。然后将6个纯化后的重组蛋白与莱姆病血清、健康人血清、梅毒血清抗体进行ELISA检测。结果利用统计学软件制作ROC曲线并进行分析,评价各个重组蛋白的灵敏度和特异性。然后将所有重组蛋白的ELISA检测结果放入logistic回归模型内进行评价,得到有诊断意义的抗原蛋白。选择莱姆病螺旋体的特异性基因recA基因为目的基因用于RPA扩增。设计了多对引物用于Basic RPA引物筛选,筛选出最合适的一对引物。根据BasicRPA筛选出的引物以及recA的基因序列设计中间探针,用于侧流层析RPA反应。产物可直接通过侧流层析试剂条反应,肉眼观察结果。对侧流层析RPA技术的最佳反应时间以及最佳反应温度进行探索,并评价其灵敏度和特异性。收集20份莱姆病病人血清,用侧流层析RPA、巢氏PCR、real time PCR分别对这20份病人血清进行检测,利用卡方检验对三种检测方法的结果进行比较,评价侧流层析RPA技术的诊断意义。经各个重组蛋白的血清学结果的ROC曲线分析,结果显示,Fla B.g在血清1gG和1gM上有较好的诊断意义(曲线下面积分别为IgG AUC=0.800, IgM AUC=0.849),但同时Fla B.g在梅毒血清诊断上具备较高的阳性率,分别为IgG 89.9%, IgM 31.4%,特异性较差。VlsE B.a的诊断血清1gG的ROC曲线下面积仅有0.736,但是其在梅毒血清诊断上阳性率最低12.3%,低于其他蛋白(P值均小于0.05)。在血清IgM诊断上,B.g型OspC诊断价值最高(AUC=0.871)。通过logistic回归模型筛选得到两个血清IgG和两个IgM诊断抗原,分别为OspC B.algM、 OspC B.g IgM、 OspC B.g IgG、VlsE B.aIgG。筛选后的模型内抗原诊断价值得到了一定的提高。通过模型模拟抗原间交互作用,发现Fla B.g与OspC B.a, Fla B.g与OspC B.g, OspC B.g与P39 B.g间存在交互作用对模型存在影响。将这三组蛋白混合后重新进行ELISA试验。结果显示OspC B.a与Fla B.g两蛋白混合不仅没有提高血清诊断的效能,反而在特异度上有所降低,降低了诊断效能。成功建立了基于侧流层析RPA的莱姆病螺旋体核酸检测技术。特异性试验体现本实验设计的方法在伯氏疏螺旋体上有特异性扩增,在与大肠埃希菌和其他非莱姆病螺旋体上无非特异扩增现象。而且该方法的最低检测下限低达50龟。三种方法检测莱姆病人血清检测后,结果显示侧流层析RPA的方法不仅在检出率上高于巢氏PCR (P0.05),与real-time PCR有相近的检出率(P0.05),而且其还具备反应时间短、方便快捷等优势。综上所述,本研究成功得到了6个纯化的重组蛋白。通过统计学方法筛评价得出了三个有诊断学价值的抗原蛋白用于莱姆病血清学抗体诊断,这三个抗原分别为OspC B.g、OspC B.a和VlsE B.a。另外OspC B.a与Fla B.g两抗原蛋白间的相互作用对莱姆病血清学诊断的影响在日后的研究中需要得到关注。另外,本研究还实验成功的建立了结合侧流层析的重组酶聚合酶扩增法用于莱姆病螺旋体的检测。该方法拥有较高的灵敏度和特异性,在30分钟时间内得到肉眼可见的结果,快速而且简便。该方法的建立尤其适用于临床床旁试验的核酸快速诊断检测。
[Abstract]:Lyme disease is a tick borne zoonosis caused by Borrelia burgdorferi. The diagnosis of Lyme disease mainly depends on clinical symptoms and laboratory assisted diagnosis. Laboratory diagnosis is particularly important in the case of unmarked clinical symptoms. However, there are no serological diagnostic reagents suitable for the standardization of the domestic leiham disease in the domestic market. The purpose of this study is to restructure and express the specific antigen of Lyme disease spirals and to evaluate its serological diagnosis. The most suitable diagnostic protein antigen is screened by statistical method, which is an immunological reagent for Lyme disease in China in the future. In addition, a new method for rapid detection of Lyme disease spiral nucleic acid based on recombinant polymerase chain amplification technique, combined with side flow chromatography, is more simple and convenient and is more conducive to the rapid diagnosis of Lyme disease. We have selected 5 kinds of protein antigens, Fla B.g (Fla central region protein), OspC (B.g and B. a), P39 B.g, P83 -E4 B.g and VlsE B.a were cloned and purified. The recombinant protein was detected by immunoblot test. The recombinant protein after 6 purified recombinant proteins was detected with Lyme disease serum, healthy human serum and syphilis sera antibody by ELISA. Results the ROC curve was made and analyzed by using the system software. The sensitivity and specificity of each recombinant protein. Then the ELISA detection results of all the recombinant proteins were put into the logistic regression model for evaluation, and the diagnostic antigen protein was obtained. The specific gene recA gene of Lyme disease spirals was selected as the target gene for RPA amplification. A number of primers were designed for Basic RPA primer sieves. Select the most suitable pair of primers. Based on the primers selected by BasicRPA and the sequence of recA gene sequences, the intermediate probe is designed for lateral flow chromatography RPA reaction. The product can be directly observed by side flow chromatography reagents and the naked eye results. The best reaction time and optimum reaction temperature of the side flow chromatography RPA are explored, and the evaluation of the best reaction temperature is also evaluated. Price sensitivity and specificity. 20 serums of Lyme disease patients were collected by side flow chromatography RPA, nesting PCR and real time PCR respectively. The results of the three detection methods were compared with the chi square test, and the diagnostic meaning of the side flow chromatography RPA technique was evaluated. The ROC curve of the serological results of each recombinant protein was measured. The results of line analysis showed that Fla B.g had a good diagnostic value on serum 1gG and 1gM (the area under the curve was IgG AUC=0.800, IgM AUC=0.849). But at the same time, Fla B.g had a higher positive rate in the diagnosis of syphilis sera, respectively, IgG 89.9%, IgM 31.4%, and only 0.736 of the diagnostic serum of poor specificity. But the positive rate in the diagnosis of syphilis serum was 12.3%, lower than that of other proteins (P value was less than 0.05). In the diagnosis of IgM, the diagnostic value of B.g type OspC was the highest (AUC=0.871). Two serum IgG and two IgM diagnostic antigens were screened by logistic regression model, respectively, OspC B.algM, OspC B.g. Fla B.g and OspC B.a, Fla B.g and OspC B.g, and the interaction between OspC B.g and P39 B.g were found to be affected by the interaction between the models, and the interaction between OspC B.g and P39 B.g was found to be influenced by the interaction between the OspC B.g and P39 B.g. The mixture not only did not improve the diagnostic efficiency of serum, but decreased the specificity, and reduced the diagnostic efficiency. The detection technique of Lyme disease spiral nucleic acid based on lateral flow chromatography RPA was successfully established. There was no specific amplification in Lyme disease spirals. And the lowest detection limit of the method was as low as 50 tortoises. Three methods detected the serum test of lym patients. The results showed that the method of lateral flow chromatography RPA was not only higher than nesting PCR (P0.05), but similar to real-time PCR (P0.05), but also had a short reaction time. In summary, 6 purified recombinant proteins were successfully obtained. Through statistical screening, three diagnostic antigens with diagnostic values were used for Lyme serological antibody diagnosis. The three antigens were OspC B.g, OspC B.a and VlsE B.a., and OspC B.a and Fla B.g two antigen protein. The influence of interaction on Lyme disease serological diagnosis needs to be paid attention in the future research. In addition, this study has also successfully established a recombinant enzyme polymerase amplification method combined with lateral flow chromatography for the detection of Lyme disease spirals. This method has high sensitivity and specificity, and is visible to the naked eye within 30 minutes. The established method is especially suitable for rapid nucleic acid diagnosis in clinical bedside tests.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R514;R446.6

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