本文選題：流感病毒 + H1N1��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年碩士論文

【摘要】：流感病毒長(zhǎng)期威脅人類(lèi)健康，其中甲型流感病毒每年都會(huì)出現(xiàn)并不斷變異，有時(shí)甚至?xí)䦟?dǎo)致流感疫情大規(guī)模爆發(fā)流行，而防控的關(guān)鍵環(huán)節(jié)之一便是通過(guò)檢測(cè)病原體對(duì)流感疑似病例的快速篩查和診斷�，F(xiàn)有的病毒分離培養(yǎng)、酶聯(lián)免疫吸附（ELISA）法以及熒光定量PCR等檢測(cè)方法在疫情檢測(cè)中發(fā)揮了重要作用，但在時(shí)效性、靈敏度和操作簡(jiǎn)便性方面仍存在不足，不能滿(mǎn)足病原體現(xiàn)場(chǎng)快速檢測(cè)的需求。 針對(duì)上述需求，本論文以A/Beijing/501/2009甲型H1N1流感病毒為檢測(cè)對(duì)象，于一次性絲網(wǎng)印刷碳電極（SPCE）上構(gòu)建了甲型H1N1流感病毒電化學(xué)免疫生物傳感器（EIS），探索了流感病毒的電化學(xué)免疫傳感分析檢測(cè)的相關(guān)機(jī)制，建立了甲型H1N1流感病毒電化學(xué)免疫生物傳感檢測(cè)技術(shù)和方法，主要研究結(jié)果如下： （1）對(duì)實(shí)驗(yàn)研究所需的材料，如流感病毒和相應(yīng)的單克隆抗體和HRP標(biāo)記抗體進(jìn)行了培養(yǎng)和收集，并對(duì)這些材料進(jìn)行了驗(yàn)證。用WHO推薦引物經(jīng)RT-PCR驗(yàn)證以及流感病毒AB分型檢測(cè)膠體金試紙條檢測(cè)，結(jié)果表明培養(yǎng)的A/Beijing/501/2009（H1N1）雞胚尿囊液確屬甲型H1N1型流感病毒，血凝實(shí)驗(yàn)測(cè)定其病毒滴度為128HA Unit，證明所培養(yǎng)的A/Beijing/501/2009（H1N1）雞胚尿囊液可以用于后續(xù)的實(shí)驗(yàn)研究。同時(shí)還通過(guò)ELISA實(shí)驗(yàn)考察了所收集到的單克隆抗體A1-24和HRP標(biāo)記單抗A1-6-HRP分別與甲型H1N1型流感病毒的親和力，結(jié)果表明這兩種抗體材料能與甲型H1N1流感病毒形成親和力較高的免疫夾心復(fù)合物，該模式可以用于構(gòu)建甲型H1N1流感病毒電化學(xué)免疫傳感器。 （2）考察比較了由不同修飾材料和方法修飾電極的電化學(xué)特點(diǎn)，發(fā)現(xiàn)捕獲抗體經(jīng)物理吸附法固定修飾電極后具有較高的信號(hào)/本底值比、操作步驟簡(jiǎn)單且不需要使用價(jià)格昂貴的納米材料或其他化學(xué)修飾材料，成本低廉，故最終選擇此方法作為捕獲抗體修飾固定電極的方法，并以此構(gòu)建甲型H1N1流感病毒電化學(xué)免疫傳感器。通過(guò)循環(huán)伏安法對(duì)該型免疫傳感器的電化學(xué)行為進(jìn)行了表征，結(jié)果表明該傳感器表面的電活性物質(zhì)主要通過(guò)擴(kuò)散傳質(zhì)方式產(chǎn)生電信號(hào)，在傳感器上產(chǎn)生的電流響應(yīng)信號(hào)與電極上加入的被測(cè)抗原的濃度呈正比關(guān)系，適用于對(duì)被測(cè)抗原進(jìn)行定量測(cè)定。 （3）優(yōu)化了影響傳感器檢測(cè)性能的主要工作條件：檢測(cè)中捕獲抗體H1-24和檢測(cè)抗體A1-6-HRP的濃度分別采用400μg/ml和80μg/ml，在此濃度條件下檢測(cè)信號(hào)都比較強(qiáng)，均能達(dá)到最大信號(hào)值的80%以上，能夠?qū)崿F(xiàn)對(duì)A(H1N1)流感病毒的靈敏檢測(cè)。孵育最佳溫度均為37℃，在此溫度下抗原抗體反應(yīng)的活性最高，反應(yīng)能快速達(dá)到平衡狀態(tài)，有利于提高反應(yīng)效率�？乖皺z測(cè)抗體的最佳孵育時(shí)間分別為45min和30min，在以上孵育時(shí)間條件下，檢測(cè)的靈敏度均較高，經(jīng)過(guò)優(yōu)化后該免疫傳感器的檢出時(shí)間縮短至2h以?xún)?nèi)，比傳統(tǒng)的ELISA法整體測(cè)定時(shí)間減少了1~2h，利用這種新型傳感器能夠?qū)崿F(xiàn)對(duì)A(H1N1)流感病毒的快速檢測(cè)。 （4）建立了電化學(xué)免疫傳感器測(cè)定甲型H1N1流感病毒的標(biāo)準(zhǔn)曲線，以及測(cè)定甲型H1N1流感病毒相應(yīng)的酶聯(lián)免疫吸附法（ELISA）標(biāo)準(zhǔn)曲線和熒光定量法（qPCR）標(biāo)準(zhǔn)曲線，用于對(duì)傳感器各項(xiàng)檢測(cè)性能指標(biāo)進(jìn)行考察。針對(duì)A(H1N1)流感病毒的檢測(cè)信號(hào)與針對(duì)甲型H7N9、H3N2、B型流感病毒、EV71以及腺病毒等陰性對(duì)照的信號(hào)的統(tǒng)計(jì)分析檢驗(yàn)結(jié)果表明，該傳感器的特異性良好。重復(fù)性測(cè)定結(jié)果顯示該傳感器的RSD10%，回收率為80%~110%，呈現(xiàn)出較高的準(zhǔn)確度和精密度。穩(wěn)定性實(shí)驗(yàn)結(jié)果顯示該傳感器儲(chǔ)存10天內(nèi)檢測(cè)信號(hào)幾乎不下降，30天后的測(cè)定值也能保持在85%以上，具有較好的存儲(chǔ)穩(wěn)定性。EIS法的檢測(cè)下限可達(dá)0.41HA Unit，與ELISA法相當(dāng)，并能對(duì)4~64HA Unit范圍內(nèi)的甲型H1N1流感病毒實(shí)現(xiàn)定量測(cè)定，，且EIS的總體假陽(yáng)性率為10.0%，優(yōu)于ELISA法。所建立的EIS法與qPCR的測(cè)定結(jié)果的相關(guān)系數(shù)為0.9517，說(shuō)明EIS法定量的準(zhǔn)確度能達(dá)到與qPCR一致的水平。 （5）最后應(yīng)用微型化的Emstat電化學(xué)檢測(cè)設(shè)備，基于便攜的陣列化電極以甲型H1N1流感病毒電化學(xué)免疫生物傳感器檢測(cè)的技術(shù)和方法為模型，探索了多種流感病毒的陣列化檢測(cè)，結(jié)果表明該傳感器具有推廣應(yīng)用于多種病原體同時(shí)檢測(cè)的前景，且其小型化的檢測(cè)儀器設(shè)備便于現(xiàn)場(chǎng)攜帶。
[Abstract]:Influenza viruses are threatening human health for a long time. Influenza A virus appears and mutates continuously every year. Sometimes it may even lead to a large-scale outbreak of influenza. One of the key links of prevention and control is rapid screening and diagnosis of suspected influenza cases by detection of pathogens. Existing virus isolation and culture, enzyme linked immunosorbent assay (ELISA) and fluorescence quantitative PCR detection methods have played an important role in the detection of epidemic situation, but there are still shortcomings in the aspects of timeliness, sensitivity and ease of operation, which can not meet the needs of rapid detection of pathogens in the field.
In order to meet the above requirements, the A/Beijing/501/2009 influenza A virus (H1N1) virus was used as the test object to construct an electrochemical immunosensor of influenza A virus (EIS) of influenza A virus (H1N1) on a single screen printed carbon electrode (SPCE). The related mechanism of electrochemical immunoassay for influenza virus was explored, and a influenza A (H1N1) disease was established. The main findings are as follows:
(1) the materials needed for the experimental study, such as influenza virus and the corresponding monoclonal antibody and HRP labeled antibody, were cultured and collected, and the materials were verified. The WHO recommended primers were tested by RT-PCR and the detection of colloidal gold test paper for influenza virus AB typing, and the results showed that the cultured A/Beijing/501/2009 (H1N1) chicken embryo urine was cultured. The bursa solution is a type A influenza A virus (H1N1), and its virus titer is 128HA Unit by hemagglutination test. It is proved that the cultured A/Beijing/501/2009 (H1N1) chicken embryo sac can be used for subsequent experimental study. At the same time, the monoclonal antibody A1-24 and HRP labeled monoclonal antibody A1-6-HRP respectively and a type a H1N1 type flow are also investigated by the ELISA experiment. The affinity of the virus has shown that these two antibody materials can form a high affinity complex with the H1N1 influenza A virus, which can be used to construct an electrochemical immunosensor for influenza A virus (H1N1) virus.
(2) the electrochemical characteristics of modified electrodes modified by different modified materials and methods are compared. It is found that the captured antibody has high signal / base ratio, and the operation procedure is simple and does not need to use expensive nano materials or other chemical modification materials after the modified electrode is fixed by physical adsorption method. Therefore, the cost is low, so this method is finally chosen. The electrochemical immunosensor of a influenza A virus (H1N1) was constructed as a method of capturing a fixed electrode with an antibody modified. The electrochemical behavior of the immunosensor was characterized by cyclic voltammetry. The results showed that the electroactive substance on the surface of the sensor produced electrical signals mainly through the spread mass transfer mode and produced on the sensor. The current response signal is proportional to the concentration of the tested antigen added on the electrode, and is suitable for the quantitative determination of the tested antigen.
(3) the main working conditions that affect the detection performance of the sensor are optimized: the concentration of the capture antibody H1-24 and the detection antibody A1-6-HRP, respectively, are 400 g/ml and 80 mu g/ml respectively. Under this concentration, the detection signals are all strong and can reach more than 80% of the maximum signal value, and can reach the sensitive detection of the A (H1N1) influenza virus. The best temperature of the antigen and antibody reaction at this temperature is the highest, the reaction can quickly reach the equilibrium state and is beneficial to the improvement of the reaction efficiency. The best incubation time of the antigen and detection antibody is 45min and 30min respectively. The sensitivity of the detection is high under the conditions of the incubation time above, and the immune sensor is detected after the optimization. The time is less than 2h, and 1~2h is reduced than the traditional ELISA method. The new sensor can be used to detect the A (H1N1) influenza virus quickly.
(4) the standard curve for the determination of influenza A virus (H1N1) by electrochemical immunosensor, and the standard curve of ELISA and the standard curve of fluorescence quantitative method (qPCR) for the determination of influenza A virus (H1N1), are used to investigate the detection performance of the sensor. The detection signal for the A (H1N1) virus and the detection signal of influenza virus (H1N1) virus are used. The statistical analysis of the negative control signals of influenza A (H7N9), H3N2, B influenza virus, EV71 and adenovirus showed that the specificity of the sensor was good. The reproducibility results showed that the sensor's RSD10%, the recovery rate was 80%~110%, showed high accuracy and precision. The stability test results showed that the sensor was stored in the sensor. Within 10 days, the detection signal was almost no decrease, and the measured value of 30 days later could be kept above 85%. The lower detection limit of.EIS method with better storage stability can reach 0.41HA Unit, which is equivalent to the ELISA method and can be quantified in the 4~64HA Unit range of a H1N1 virus, and the total false positive rate of EIS is 10%, superior to the ELISA method. The correlation coefficient between the established EIS method and the qPCR test result is 0.9517, indicating that the accuracy of EIS method can reach the level consistent with qPCR.
(5) at last, the microminiature Emstat electrochemical detection equipment was used to explore the array detection of a variety of influenza viruses based on the technique and method of portable array electrode based on the detection of electrochemical immunosensors of influenza A virus (H1N1). The results show that the sensor has a wide application to the simultaneous detection of a variety of pathogens. Scene, and its miniaturized testing equipment is easy to carry on the spot.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R446.6;R373

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