發(fā)布時間：2018-07-09 22:40

  本文選題：化學(xué)發(fā)光法 + 熒光法��； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：化學(xué)發(fā)光法(Chemiluminescence,CL)和熒光法(Fluorescence,FL)都屬于分子發(fā)光分析法。CL不需要激發(fā)光源,因此具有設(shè)備簡單,背景信號低,靈敏度高和線性范圍寬等優(yōu)點。FL儀器的激發(fā)光源和檢測器不在一條直線上,所以具有信噪比高、檢測限低等優(yōu)點。CL和FL結(jié)合分子識別試劑偶聯(lián)磁性微�？梢杂行У睾喕疤幚磉^程和消除基質(zhì)干擾,還可以進一步提高檢測靈敏度和準確度,非常適用于金黃色葡萄球菌(Staphylococcus aureus,S.aureus)的檢測。本文利用分子識別試劑免疫球蛋白G(Immunoglobulin G,Ig G)和噬菌體肽(Phage-displayed peptide,PDP)對金黃色葡萄球菌的特異性識別作用,結(jié)合磁分離技術(shù)分別建立了金黃色葡萄球菌的競爭化學(xué)發(fā)光檢測法和夾心熒光檢測法:(1)基于金黃色葡萄球菌表面A蛋白(Staphylococcus Protein A,SPA)偶聯(lián)磁珠與金黃色葡萄球菌競爭結(jié)合Ig G的超靈敏CL檢測金黃色葡萄球菌本研究基于SPA與Ig G結(jié)合作用,構(gòu)建競爭式CL,直接檢測金黃色葡萄球菌。SPA可以與Ig G的Fc片段結(jié)合。SPA存在于大多數(shù)的金黃色葡萄球菌的表面,而在其它細菌的表面不存在或是存在的量非常少,使得金黃色葡萄球菌與Ig G的結(jié)合具有一定的特異性。表面偶聯(lián)SPA的磁珠與金黃色葡萄球菌兩者共同競爭辣根過氧化物酶(Horseradish peroxidase,HRP)標記的Ig G(HRP-tagged Ig G)。磁分離之后,在固相中加入共反應(yīng)試劑魯米諾和過氧化氫,測定化學(xué)發(fā)光信號。實驗結(jié)果表明在最優(yōu)條件下,金黃色葡萄球菌的濃度在1.0×10~1-1.0×10~9 cfu m L~(-1)范圍內(nèi)與化學(xué)發(fā)光強度呈現(xiàn)良好的線性關(guān)系,檢測限是6.0 cfu m L~(-1)(信噪比為3)。該方法具有快速檢測、特異性好、靈敏度高、價格低廉、易于操作等優(yōu)點,有望應(yīng)用于臨床檢測、藥品安全、食品安全和環(huán)境監(jiān)測等領(lǐng)域。(2)基于雙肽識別模式的夾心FL特異性檢測金黃色葡萄球菌本研究利用兩種多肽構(gòu)建夾心FL,直接特異性檢測金黃色葡萄球菌。我們選用噬菌體肽作為目標菌的特異性識別試劑,并用異硫氰酸熒光素(Fluorescein isothiocyanate,FITC)修飾的抗菌肽magainin I(FITC-tagged magainin I)制備高靈敏的熒光探針。同時,利用噬菌體肽偶聯(lián)磁性微粒從樣品中分離、捕獲和富集金黃色葡萄球菌,從而減少基質(zhì)對熒光檢測的干擾并且提高檢測靈敏度。與噬菌體、抗體相比,噬菌體肽和抗菌肽具有性狀穩(wěn)定、價格低廉、易于合成和修飾等優(yōu)點。實驗結(jié)果表明金黃色葡萄球菌的濃度在1.0×10~1-1.0×10~5 cfu m L~(-1)范圍內(nèi)與熒光信號呈現(xiàn)良好的線性關(guān)系,檢測限為9.0 cfu m L~(-1)(信噪比為3)。該方法可以通過替換特異性噬菌體肽,實現(xiàn)對其它病原菌的檢測。
[Abstract]:Chemiluminescence method (CL) and fluorescence method (FL) both belong to molecular luminescence analysis. CL does not need excitation light source, so it has simple equipment and low background signal. High sensitivity and wide linear range. The excitation light source and detector of FL instrument are not in a straight line, so they have high signal-to-noise ratio (SNR). The advantages of low detection limit. CL and FL combined with molecular recognition reagent coupling magnetic particles can effectively simplify the pretreatment process and eliminate matrix interference, and can further improve the detection sensitivity and accuracy. It is very suitable for the detection of Staphylococcus aureus S.aureus. The specific recognition effects of immunoglobulin G (Ig G) and phage displayed peptide (PDP) on Staphylococcus aureus were studied. In combination with magnetic separation technique, competitive chemiluminescence detection and sandwich fluorescence detection of Staphylococcus aureus were established: (1) based on Staphylococcus Protein A SPA coupling magnetic beads and Staphylococcus aureus competitive knot Detection of Staphylococcus aureus by hypersensitive CL combined with Ig G this study was based on the binding effect of SPA and Ig G. Competitive CLs were constructed to directly detect the presence of S.aureus SPA on the surface of most Staphylococcus aureus with the FC fragment of Ig G, but not on the surface of other bacteria or in very small quantities. The binding of Staphylococcus aureus to IgG has a certain specificity. The surface coupled magnetic beads of SPA and Staphylococcus aureus competed with horseradish peroxidase (HRP) labeled IgG (HRP-tagged Ig G). After magnetic separation, the chemiluminescence signals were determined by the addition of the co-reaction reagents Luminol and hydrogen peroxide in the solid phase. The experimental results showed that the concentration of Staphylococcus aureus in the range of 1.0 脳 10 ~ (-1) ~ 1.0 脳 10 ~ (9) cfu mL ~ (-1) showed a good linear relationship with the chemiluminescence intensity under the optimum conditions, and the detection limit was 6.0 cfu mL ~ (-1) (SNR 3). The method has the advantages of rapid detection, good specificity, high sensitivity, low price and easy operation. It is expected to be used in clinical detection and drug safety. Food safety and environmental monitoring. (2) FL-specific detection of Staphylococcus aureus based on double peptide recognition pattern. In this study, two peptides were used to construct sandwich FLL to detect Staphylococcus aureus directly. The bacteriophage peptide was selected as the specific recognition reagent of the target strain, and a highly sensitive fluorescent probe was prepared with fluororescein isothiocyanate FITC modified antimicrobial peptide magainin I (FITC-tagged magainin I). At the same time, the phage peptide coupled magnetic particles were used to isolate Staphylococcus aureus from the sample to capture and enrich Staphylococcus aureus so as to reduce the interference of the matrix to the fluorescence detection and improve the detection sensitivity. Compared with phage and antibody, bacteriophage peptide and antimicrobial peptide have the advantages of stable character, low price, easy synthesis and modification. The results showed that the concentration of Staphylococcus aureus in the range of 1.0 脳 10 ~ (-1) ~ 1.0 脳 10 ~ (5) cfu mL ~ (-1) showed a good linear relationship with the fluorescence signal, and the detection limit was 9.0 cfu mL ~ (-1) (SNR 3). This method can be used to detect other pathogens by replacing specific phage peptides.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O657.3;R446.5

【相似文獻】

相關(guān)期刊論文 前10條

1 吳雅兒;橙汁飲料中金黃色葡萄球菌的檢測與研究[J];職業(yè)與健康;2000年11期

2 白潔;機組配餐員的金黃色葡萄球菌污染情況[J];國外醫(yī)學(xué)(衛(wèi)生學(xué)分冊);2002年02期

3 朱自強;;金黃色葡萄球菌更具殺傷力的原因在于其金黃色“外殼”[J];基礎(chǔ)醫(yī)學(xué)與臨床;2005年11期

4 杜洪利;張雙雙;歐旭;閆訓(xùn)友;;金黃色葡萄球菌檢測技術(shù)研究進展[J];食品與發(fā)酵科技;2009年05期

5 黃嶺芳;賴衛(wèi)華;張莉莉;;食品中金黃色葡萄球菌快速檢測方法的研究進展[J];食品與機械;2009年06期

6 王娉;勞華均;胡s，

本文編號：2110951