基于分子识别模式和磁分离的化学发光与荧光法特异性检测金黄色葡萄球菌

发布时间：2018-07-09 22:40

本文选题：化学发光法 + 荧光法　；参考：《西南大学》2017年硕士论文

【摘要】：化学发光法(Chemiluminescence,CL)和荧光法(Fluorescence,FL)都属于分子发光分析法。CL不需要激发光源,因此具有设备简单,背景信号低,灵敏度高和线性范围宽等优点。FL仪器的激发光源和检测器不在一条直线上,所以具有信噪比高、检测限低等优点。CL和FL结合分子识别试剂偶联磁性微粒可以有效地简化前处理过程和消除基质干扰,还可以进一步提高检测灵敏度和准确度,非常适用于金黄色葡萄球菌(Staphylococcus aureus,S.aureus)的检测。本文利用分子识别试剂免疫球蛋白G(Immunoglobulin G,Ig G)和噬菌体肽(Phage-displayed peptide,PDP)对金黄色葡萄球菌的特异性识别作用,结合磁分离技术分别建立了金黄色葡萄球菌的竞争化学发光检测法和夹心荧光检测法:(1)基于金黄色葡萄球菌表面A蛋白(Staphylococcus Protein A,SPA)偶联磁珠与金黄色葡萄球菌竞争结合Ig G的超灵敏CL检测金黄色葡萄球菌本研究基于SPA与Ig G结合作用,构建竞争式CL,直接检测金黄色葡萄球菌。SPA可以与Ig G的Fc片段结合。SPA存在于大多数的金黄色葡萄球菌的表面,而在其它细菌的表面不存在或是存在的量非常少,使得金黄色葡萄球菌与Ig G的结合具有一定的特异性。表面偶联SPA的磁珠与金黄色葡萄球菌两者共同竞争辣根过氧化物酶(Horseradish peroxidase,HRP)标记的Ig G(HRP-tagged Ig G)。磁分离之后,在固相中加入共反应试剂鲁米诺和过氧化氢,测定化学发光信号。实验结果表明在最优条件下,金黄色葡萄球菌的浓度在1.0×10~1-1.0×10~9 cfu m L~(-1)范围内与化学发光强度呈现良好的线性关系,检测限是6.0 cfu m L~(-1)(信噪比为3)。该方法具有快速检测、特异性好、灵敏度高、价格低廉、易于操作等优点,有望应用于临床检测、药品安全、食品安全和环境监测等领域。(2)基于双肽识别模式的夹心FL特异性检测金黄色葡萄球菌本研究利用两种多肽构建夹心FL,直接特异性检测金黄色葡萄球菌。我们选用噬菌体肽作为目标菌的特异性识别试剂,并用异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)修饰的抗菌肽magainin I(FITC-tagged magainin I)制备高灵敏的荧光探针。同时,利用噬菌体肽偶联磁性微粒从样品中分离、捕获和富集金黄色葡萄球菌,从而减少基质对荧光检测的干扰并且提高检测灵敏度。与噬菌体、抗体相比,噬菌体肽和抗菌肽具有性状稳定、价格低廉、易于合成和修饰等优点。实验结果表明金黄色葡萄球菌的浓度在1.0×10~1-1.0×10~5 cfu m L~(-1)范围内与荧光信号呈现良好的线性关系,检测限为9.0 cfu m L~(-1)(信噪比为3)。该方法可以通过替换特异性噬菌体肽,实现对其它病原菌的检测。
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3;R446.5

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