本文選題：萊姆病 + 伯氏疏螺旋體　； 參考：《中國(guó)疾病預(yù)防控制中心》2016年碩士論文

【摘要】：萊姆病是由伯氏疏螺旋體引起的蜱傳人獸共患病。萊姆病的診斷主要依靠臨床癥狀和實(shí)驗(yàn)室輔助診斷。在臨床癥狀不顯著的情況下,實(shí)驗(yàn)室診斷尤為重要。但是目前國(guó)內(nèi)市場(chǎng)上暫未出現(xiàn)適合于國(guó)內(nèi)萊姆病標(biāo)準(zhǔn)商品化的血清學(xué)診斷試劑盒,另外在萊姆病的快速檢測(cè)方面仍需要提高。本研究的目的主要在于重組表達(dá)萊姆病螺旋體特異性抗原并對(duì)其進(jìn)行了血清學(xué)診斷評(píng)價(jià),通過(guò)統(tǒng)計(jì)學(xué)方法篩選出最合適的有診斷意義的蛋白抗原,為日后研制中國(guó)萊姆病免疫學(xué)檢測(cè)試劑盒提供依據(jù)。另外建立一種新的基于重組酶聚合酶擴(kuò)增技術(shù)的萊姆病螺旋體核酸快速檢測(cè)方法,結(jié)合側(cè)流層析技術(shù),更簡(jiǎn)單方便,更有利于萊姆病的快速診斷。我們共選取了5種蛋白抗原,Fla B.g (Fla中央?yún)^(qū)蛋白),OspC (B.g型和B.a型),P39 B.g, P83-E4 B.g和VlsE B.a,對(duì)其進(jìn)行了克隆表達(dá)和純化。利用免疫印跡實(shí)驗(yàn)對(duì)重組蛋白進(jìn)行初步免疫原性檢測(cè)。然后將6個(gè)純化后的重組蛋白與萊姆病血清、健康人血清、梅毒血清抗體進(jìn)行ELISA檢測(cè)。結(jié)果利用統(tǒng)計(jì)學(xué)軟件制作ROC曲線并進(jìn)行分析,評(píng)價(jià)各個(gè)重組蛋白的靈敏度和特異性。然后將所有重組蛋白的ELISA檢測(cè)結(jié)果放入logistic回歸模型內(nèi)進(jìn)行評(píng)價(jià),得到有診斷意義的抗原蛋白。選擇萊姆病螺旋體的特異性基因recA基因?yàn)槟康幕蛴糜赗PA擴(kuò)增。設(shè)計(jì)了多對(duì)引物用于Basic RPA引物篩選,篩選出最合適的一對(duì)引物。根據(jù)BasicRPA篩選出的引物以及recA的基因序列設(shè)計(jì)中間探針,用于側(cè)流層析RPA反應(yīng)。產(chǎn)物可直接通過(guò)側(cè)流層析試劑條反應(yīng),肉眼觀察結(jié)果。對(duì)側(cè)流層析RPA技術(shù)的最佳反應(yīng)時(shí)間以及最佳反應(yīng)溫度進(jìn)行探索,并評(píng)價(jià)其靈敏度和特異性。收集20份萊姆病病人血清,用側(cè)流層析RPA、巢氏PCR、real time PCR分別對(duì)這20份病人血清進(jìn)行檢測(cè),利用卡方檢驗(yàn)對(duì)三種檢測(cè)方法的結(jié)果進(jìn)行比較,評(píng)價(jià)側(cè)流層析RPA技術(shù)的診斷意義。經(jīng)各個(gè)重組蛋白的血清學(xué)結(jié)果的ROC曲線分析,結(jié)果顯示,Fla B.g在血清1gG和1gM上有較好的診斷意義(曲線下面積分別為IgG AUC=0.800, IgM AUC=0.849),但同時(shí)Fla B.g在梅毒血清診斷上具備較高的陽(yáng)性率,分別為IgG 89.9%, IgM 31.4%,特異性較差。VlsE B.a的診斷血清1gG的ROC曲線下面積僅有0.736,但是其在梅毒血清診斷上陽(yáng)性率最低12.3%,低于其他蛋白(P值均小于0.05)。在血清IgM診斷上,B.g型OspC診斷價(jià)值最高(AUC=0.871)。通過(guò)logistic回歸模型篩選得到兩個(gè)血清IgG和兩個(gè)IgM診斷抗原,分別為OspC B.algM、 OspC B.g IgM、 OspC B.g IgG、VlsE B.aIgG。篩選后的模型內(nèi)抗原診斷價(jià)值得到了一定的提高。通過(guò)模型模擬抗原間交互作用,發(fā)現(xiàn)Fla B.g與OspC B.a, Fla B.g與OspC B.g, OspC B.g與P39 B.g間存在交互作用對(duì)模型存在影響。將這三組蛋白混合后重新進(jìn)行ELISA試驗(yàn)。結(jié)果顯示OspC B.a與Fla B.g兩蛋白混合不僅沒(méi)有提高血清診斷的效能,反而在特異度上有所降低,降低了診斷效能。成功建立了基于側(cè)流層析RPA的萊姆病螺旋體核酸檢測(cè)技術(shù)。特異性試驗(yàn)體現(xiàn)本實(shí)驗(yàn)設(shè)計(jì)的方法在伯氏疏螺旋體上有特異性擴(kuò)增,在與大腸埃希菌和其他非萊姆病螺旋體上無(wú)非特異擴(kuò)增現(xiàn)象。而且該方法的最低檢測(cè)下限低達(dá)50龜。三種方法檢測(cè)萊姆病人血清檢測(cè)后,結(jié)果顯示側(cè)流層析RPA的方法不僅在檢出率上高于巢氏PCR (P0.05),與real-time PCR有相近的檢出率(P0.05),而且其還具備反應(yīng)時(shí)間短、方便快捷等優(yōu)勢(shì)。綜上所述,本研究成功得到了6個(gè)純化的重組蛋白。通過(guò)統(tǒng)計(jì)學(xué)方法篩評(píng)價(jià)得出了三個(gè)有診斷學(xué)價(jià)值的抗原蛋白用于萊姆病血清學(xué)抗體診斷,這三個(gè)抗原分別為OspC B.g、OspC B.a和VlsE B.a。另外OspC B.a與Fla B.g兩抗原蛋白間的相互作用對(duì)萊姆病血清學(xué)診斷的影響在日后的研究中需要得到關(guān)注。另外,本研究還實(shí)驗(yàn)成功的建立了結(jié)合側(cè)流層析的重組酶聚合酶擴(kuò)增法用于萊姆病螺旋體的檢測(cè)。該方法擁有較高的靈敏度和特異性,在30分鐘時(shí)間內(nèi)得到肉眼可見(jiàn)的結(jié)果,快速而且簡(jiǎn)便。該方法的建立尤其適用于臨床床旁試驗(yàn)的核酸快速診斷檢測(cè)。
[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.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R514;R446.6

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