本文選題：腸炎沙門氏菌 + 可視化檢測(cè)��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：食品貿(mào)易作為國(guó)際貿(mào)易的主要組成部分,在擴(kuò)大出口創(chuàng)匯、解決糧食供求矛盾的同時(shí),也帶來了食源性疾病的發(fā)病率和死亡率上升的問題。微生物病原是我國(guó)食源性疾病的主要病因,占30%~40%,細(xì)菌占微生物病原的81.5%。因此,食源性疾病尤其是由細(xì)菌污染引起的食源性疾病是我國(guó)食品安全面臨的首要問題。我國(guó)作為一個(gè)食品生產(chǎn)和消費(fèi)大國(guó),建立快速、準(zhǔn)確的食源性病原菌檢測(cè)技術(shù),對(duì)于食品質(zhì)量監(jiān)控、保證人民健康至關(guān)重要。目前食源性細(xì)菌檢測(cè)的方法主要有三大類:平皿培養(yǎng)法、免疫學(xué)檢測(cè)以及現(xiàn)代分子生物學(xué)檢測(cè)方法。平皿培養(yǎng)法使用的是傳統(tǒng)的培養(yǎng)法,不需要復(fù)雜的實(shí)驗(yàn)儀器并且實(shí)驗(yàn)結(jié)果可靠,但是檢測(cè)周期較長(zhǎng),5~7天,而且程序繁瑣、費(fèi)時(shí)費(fèi)力。經(jīng)典的酶連免疫吸附技術(shù)(ELISA)等免疫學(xué)檢測(cè)方法,特異性高、靈敏性高,但是需要多種蛋白質(zhì)分子(如單克隆抗體)的參與,成本較高,而且對(duì)反應(yīng)環(huán)境要求苛刻。新型免疫熒光技術(shù)也具有對(duì)試劑、儀器選擇性高的缺點(diǎn)。分子生物學(xué)方法針對(duì)的靶標(biāo)是核酸,但是由于生物體內(nèi)核酸的含量通常是十分微少的,因此經(jīng)過溫度循環(huán)的核酸擴(kuò)增如聚合酶鏈?zhǔn)椒磻?yīng)(PCR)、實(shí)時(shí)定量PCR幾乎是這類檢測(cè)方法的前提步驟。但是對(duì)于落后地區(qū)或者設(shè)備資源貧乏的環(huán)境,這些檢測(cè)方法也受到限制。雜交鏈?zhǔn)椒磻?yīng)(HCR)是一種無(wú)酶參與、室溫條件便可以進(jìn)行的核酸擴(kuò)增反應(yīng)。相對(duì)于PCR,HCR并不是對(duì)于模板序列的直接擴(kuò)增,而是間接的對(duì)核酸信號(hào)進(jìn)行放大。由于具有無(wú)酶參與,反應(yīng)條件溫和等特點(diǎn),近年來,HCR反應(yīng)與電化學(xué)、熒光信號(hào)技術(shù)結(jié)合已經(jīng)被廣泛應(yīng)用于核酸、蛋白、病原菌等靶標(biāo)物質(zhì)的檢測(cè)�？梢暬瘷z測(cè)技術(shù),是指檢測(cè)的結(jié)果可以在可見光或者紫外燈下被肉眼觀察的實(shí)驗(yàn)方法。相對(duì)于其他檢測(cè)方法,因?yàn)椴恍枰軆x器參與,因此檢測(cè)成本較低,適用場(chǎng)合較廣。本實(shí)驗(yàn)的目的是以核酸等溫?cái)U(kuò)增反應(yīng)HCR作為信號(hào)放大手段,以腸炎沙門氏菌為檢測(cè)靶標(biāo),建立快速、低成本的檢測(cè)方法。我們利用NCBI數(shù)據(jù)庫(kù)中Blast比對(duì)功能篩選出腸炎沙門氏菌核糖體16S rRNA特異性片段作為靶標(biāo),設(shè)計(jì)相應(yīng)的特異性的抓取探針、檢測(cè)探針,并以HCR反應(yīng)作為信號(hào)放大手段,分別結(jié)合膠體金免疫層析試紙條、微孔板顯色這兩種方法建立低成本、快速便捷的即時(shí)診斷技術(shù)(POCT)。在膠體金免疫層析試紙條方法中,我們首先制備了檢測(cè)試紙條:納米金顆粒上包被有鏈霉親和素、檢測(cè)線上涂有熒光基團(tuán)的抗體,質(zhì)控線上涂有生物素。然后以合成的模擬靶標(biāo)建立檢測(cè)模型,并進(jìn)行相應(yīng)的條件優(yōu)化。我們發(fā)現(xiàn)HCR反應(yīng)中最佳啟動(dòng)探針/發(fā)夾探針的濃度比例是1:5,修飾探針/不修飾探針的含量比例是9:1,最佳抓取探針濃度是0.3μM并且能夠區(qū)分靶標(biāo)沙門氏菌序列與弗氏檸檬酸桿菌、大腸桿菌、阪崎腸桿菌、金黃色葡萄球菌、耶爾森氏菌五種細(xì)菌的相似性片段。以合成序列為靶標(biāo)的檢測(cè)模型中,未結(jié)合HCR反應(yīng)的檢測(cè)限是0.31nM,結(jié)合HCR反應(yīng)的方法可以檢測(cè)到1.76pM,放大倍數(shù)為176倍。然后我們將培養(yǎng)的沙門氏菌進(jìn)行平板計(jì)數(shù)、并提取其總RNA,結(jié)合本方法進(jìn)行實(shí)際樣本的檢測(cè),計(jì)算的LOD值為3×103 CFU mL-1。在獲取RNA樣本之后,整個(gè)檢測(cè)時(shí)間在30min之內(nèi),并且結(jié)果可以用肉眼判斷。在微孔板顯色實(shí)驗(yàn)中,包被由鏈霉親和素的微孔板用于固定具有特異性識(shí)別能力的、修飾有生物素分子的抓取探針。靶標(biāo)序列的存在,使得夾心結(jié)構(gòu):抓取探針/靶標(biāo)序列/HCR得以形成,并由于生物素與鏈霉親和素之間親合作用被固定到微孔板上,由于HCR產(chǎn)物長(zhǎng)鏈上標(biāo)記有熒光基團(tuán)FITC,可以結(jié)合辣根過氧化物酶(HRP)標(biāo)記的熒光集團(tuán)抗體anti-FITC-HRP。HRP的存在不僅可以催化顯色反應(yīng),而且由于自身的高催化活性,達(dá)到雙重信號(hào)放大的能力。實(shí)驗(yàn)過程中,我們同樣的進(jìn)行了條件優(yōu)化:最佳抓取探針的濃度是0.5μM,最佳孵育時(shí)是2小時(shí),anti-FITC-HRP的最佳孵育時(shí)長(zhǎng)是2小時(shí)。本實(shí)驗(yàn)設(shè)計(jì)的探針能夠區(qū)分與靶標(biāo)有一個(gè)堿基差異的序列,以合成序列建立檢測(cè)模型的檢測(cè)限是32.6fM。然后我們將培養(yǎng)的沙門氏菌進(jìn)行平板計(jì)數(shù)、并提取其總RNA,將提取的細(xì)菌RNA用于檢測(cè),不僅可以將沙門氏菌與弗氏檸檬酸桿菌、大腸桿菌、金黃色葡萄球菌區(qū)分開(非靶標(biāo)組的信號(hào)值均不高于22%),而且針對(duì)沙門氏菌的檢測(cè)限可以達(dá)到52.1 CFU mL-1。上述結(jié)果表明,本文所建立的雜交鏈?zhǔn)椒磻?yīng)分別結(jié)合免疫層析試紙條、微孔板顯色用于沙門氏菌可視化檢測(cè)的方法,具有良好的特異性以及靈敏度。相對(duì)于運(yùn)用電化學(xué)、熒光等技術(shù)結(jié)合HCR反應(yīng)的檢測(cè)方法,本文所介紹的兩種實(shí)驗(yàn)方法在檢測(cè)的靈敏性方面并不是最佳的,但是由于本實(shí)驗(yàn)所運(yùn)用的膠體金免疫層析試紙條、微孔板顯色方法的實(shí)驗(yàn)結(jié)果都可以用肉眼判斷,不需要高端儀器的配合,并且實(shí)驗(yàn)過程簡(jiǎn)單,不需要專業(yè)人士的操作,因此比較適用于偏遠(yuǎn)、資源貧乏的地區(qū)用于檢測(cè),可以作為POCT診斷試劑進(jìn)一步的進(jìn)行開發(fā)、應(yīng)用。
[Abstract]:As the main part of the international trade, the food trade is the main cause of food borne diseases in China, which is the main cause of food borne diseases in China, which accounts for the 30%~40%, and the microbes account for the 81.5%. of the microbial pathogens, especially the food borne diseases. The food borne disease caused by bacterial contamination is the primary problem of food safety in China. As a big food production and consumption country, China is a major food producing and consuming country. It is very important for food quality monitoring and to ensure the health of the people to establish rapid and accurate detection techniques for food borne pathogens. There are three main categories of food borne bacteria detection methods: The method of plate culture, immunology and modern molecular biology test. The plate culture method uses the traditional culture method. It does not need complex experimental instruments and the experimental results are reliable, but the detection cycle is long, 5~7 days, and the program is tedious, time-consuming and laborious. The classic enzyme linked immunosorbent assay (ELISA) and other immunological detection parties It has high specificity and high sensitivity, but requires a variety of protein molecules, such as monoclonal antibodies, with high cost and harsh response to the environment. New immunofluorescence technology also has the disadvantage of high selectivity for reagents and instruments. The target of molecular biology is nucleic acid, but the content of nucleic acid in organism is through. It is often very small, so the real-time quantitative PCR is almost the precondition of this kind of detection through the RNA amplification of temperature cycle like polymerase chain reaction (PCR). But for the backward areas or the poor environment of equipment resources, these detection methods are also restricted. The mixed chain reaction (HCR) is a kind of non enzyme participation, room temperature condition. Compared with PCR, HCR is not a direct amplification of the template sequence, but an indirect amplification of nucleic acid signals. The combination of HCR reaction with electrochemistry and fluorescence signal technology has been widely used in nucleic acid, protein, pathogen and so on in recent years. Detection of target materials. Visual detection technology means that the results of detection can be observed by the naked eye under visible light or ultraviolet light. Compared with other methods, the detection cost is low and the application is wide. The purpose of this experiment is to amplify HCR as a letter by isothermal amplification of nucleic acid. A rapid and low cost detection method was established with Salmonella enteritidis as a target test. We screened the specific fragment of the ribosome 16S rRNA specific fragment of Salmonella enteritidis as a target by using the Blast comparison function in the NCBI database, designed the specific grasping probe, detected the probe, and put the HCR reaction as a signal hand. In a colloidal gold immunochromatographic test paper method, we first prepared the test paper in the colloidal gold immunochromatographic test strip method, which was coated with streptomycin avidin on the gold nanoparticles, and the detection of the antibody on the line with fluorescent groups was detected in the two methods. The control line is coated with biotin. Then the test model is set up with the synthetic target, and the corresponding conditions are optimized. We found that the concentration ratio of the best start probe / hairpin probe in the HCR reaction is 1:5, the content ratio of the modified probe / unmodified probe is 9:1, the best grip concentration is 0.3 mu M and the target salmonk can be distinguished. The sequence is similar to the five bacteria of citric acid bacilli, Escherichia coli, Enterobacter sakazakii, Staphylococcus aureus, and Jerson S bacteria. In the detection model of the synthetic sequence as the target, the detection limit of unbound HCR reaction is 0.31nM, and the method of combining the HCR reaction can detect 1.76pM, and the magnification is 176 times. And then we will The cultured Salmonella was counted, and the total RNA was extracted, and the actual sample was detected with this method. The calculated LOD value was 3 x 103 CFU mL-1. after obtaining the RNA sample, the whole detection time was within 30min, and the result could be judged by the naked eye. The presence of the target sequence makes the sandwich structure: the capture probe / target sequence /HCR is formed, and the affinity between biotin and streptomycin is immobilized to the microporous plate because of the fluorescent group FITC marked on the long chain of the HCR product. The presence of the fluorescent group antibody anti-FITC-HRP.HRP labeled with horseradish peroxidase (HRP) not only catalyzes the color reaction, but also has the ability to achieve double signal amplification due to its high catalytic activity. In the experiment, the same conditions were optimized: the concentration of the best capture probe was 0.5 u M, and the best incubation was 2 hours. The best incubation time of anti-FITC-HRP is 2 hours. The probe designed in this experiment can distinguish the sequence of base difference between the target and the target. The detection limit of the synthetic sequence is 32.6fM., then we will count the Salmonella in the plate, and extract the total RNA, which can be used to detect the bacterial RNA. Salmonella was separated from citric acid bacilli, Escherichia coli and Staphylococcus aureus (the signal value of the non target group was not higher than 22%), and the detection limit for Salmonella could reach 52.1 CFU mL-1.. The results showed that the hybrid chain reaction established in this paper was combined with the immunochromatography test strip and the microplate was used to display color. The method of visual detection of Salmonella has good specificity and sensitivity. Compared with the application of electrochemistry, fluorescence and other techniques combined with HCR reaction, the two methods introduced in this paper are not the best in sensitivity of detection, but the colloidal gold immunochromatography paper strips used in this experiment are micro. The experimental results of the hole plate color display method can be judged by the naked eye. It does not need the cooperation of the high-end instrument, and the experiment process is simple. It does not need the operation of the professionals. Therefore, it is more suitable for remote, poor resource areas for detection. It can be developed and applied as a POCT diagnostic reagent.

【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】：2017
【分類號(hào)】：R446.5

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本文編號(hào)：1835838