食品中布鲁氏菌快速检测方法的建立及效果评价

发布时间：2018-03-16 15:35

本文选题：布鲁氏菌　切入点：多克隆抗体　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：布鲁氏菌是一种重要的人兽共患病病原菌,它的传播和流行给畜牧业及人类健康造成巨大的威胁,加强对其检测,对于布鲁氏菌病的防控具有重要的意义。现有的布鲁氏菌检测方法或多或少存在着操作复杂、耗时较长、灵敏度低以及特异性差等缺点,因此建立快速、灵敏且特异的布鲁氏菌检测方法十分必要。本课题旨在制备布鲁氏菌兔多克隆抗体,并联合免疫磁珠分离技术和量子点标记技术建立一种布鲁氏菌快速检测方法,并对该方法的检测效果进行评价。本课题的研究内容包括以下两个方面:1.布鲁氏菌兔多克隆抗体的制备及鉴定采用布鲁氏菌16M制备疫苗,免疫新西兰大白兔,制备多克隆抗体,利用饱和硫酸铵盐析法对抗体进行纯化。建立间接ELISA方法,使用矩阵法对以下反应条件进行优化,包括抗原包被浓度、血清工作浓度、包被条件、封闭液种类、封闭时间、抗原抗体反应时间、二抗工作浓度、二抗反应时间和显色时间,并用优化的间接ELISA方法测定抗体效价和其特异性;采用SDS-PAGE方法测定多克隆抗体的纯度;采用Bradford蛋白浓度测定试剂盒测定多克隆抗体的蛋白含量。结果显示,间接ELISA方法的最优条件为:最佳抗原包被条件为37℃包被1 h、浓度为1:40,最佳封闭液为5%脱脂奶粉,最佳封闭时间为1 h,最佳血清工作浓度为1:16000,最佳抗原抗体反应时间为1.5 h,最佳二抗工作浓度为1:10000,二抗反应时间为0.5 h,最佳显色时间为10 min;1号抗体效价为1:256000,2号抗体效价为1:2048000;抗体特异性较好,与大肠杆菌O157、单增李斯特菌、奇异变形杆菌、阴沟肠杆菌、坂崎肠杆菌、克雷伯杆菌、鲍氏志贺氏菌、金黄色葡萄球菌、粘质沙雷氏菌、沙门氏菌交叉反应小;抗体纯度较好;1号和2号抗体蛋白含量分别为14.091 mg/m L和17.454 mg/m L。2.检测方法的建立与评价将羧基化磁珠与制备的布鲁氏菌多克隆抗体共价偶联,制备可识别布鲁氏菌的磁纳米探针;将羧基化荧光量子点与实验室前期制备的布鲁氏菌鸡卵黄抗体(Ig Y)共价偶联,制备可以识别布鲁氏菌的量子点荧光生物探针,基于这两种生物探针建立一种新的布鲁氏菌检测方法,得到磁纳米探针—布鲁氏菌—量子点荧光生物探针“三明治”夹心复合物,利用荧光光谱仪对复合物进行荧光检测,建立荧光强度与菌液浓度的线性回归方程,从而实现对布鲁氏菌的快速、定量的检测。在检测方法建立过程中,对磁纳米探针的用量、富集时间、量子点探针的用量以及标记时间进行优化,评价检测方法的灵敏度、特异性和重复性,并用所建立的检测方法对人工模拟的肉样和牛奶样品进行检测。结果显示,磁纳米生物探针制备的最佳条件为100μL磁珠与122.178μg兔多克隆抗体于37℃偶联2 h;量子点生物探针制备的最佳条件为10μL QDs与262μg Ig Y于37℃偶联2 h。所建立的布鲁氏菌检测方法最佳反应条件为:磁纳米探针最佳用量为100μL,最佳富集时间为45 min,量子点探针最佳用量为500μL,最佳标记时间为60 min;该方法的检测时间约为105 min,反应产物的荧光强度与菌液浓度的对数呈线性关系,线性方程为y=21.763x+78.371(R2=0.9983),检测限为103 CFU/m L;特异性实验结果显示,该方法能识别羊种和猪种布鲁氏菌,但是不能识别大肠杆菌O157和单增李斯特菌,特异性较好;在牛奶模拟样和肉汁模拟样中的检测限均为102 CFU/m L,说明该方法在模拟样品中的检测效果较好。综上所述,本课题制备了布鲁氏菌兔多克隆抗体、能够识别布鲁氏菌的磁纳米生物探针和量子点荧光生物探针,并建立了一种基于磁性分离技术和量子点标记技术的布鲁氏菌快速检测方法。该检测方法的检测限较低,特异性较好,且具有较好的稳定性,在模拟样品的检测中也能得到良好的应用,本研究工作建立了具有我国自主知识产权的布鲁氏菌的快速检测方法。
[Abstract]:Brucellosis is an important zoonotic pathogen, its spread and epidemic caused a great threat to human health and animal husbandry, strengthen the inspection, has an important significance for the prevention and control of brucellosis Brucella. The existing detection methods are more or less complicated operation, time-consuming, low sensitivity and poor specificity shortcomings, so establishing a rapid detection method, it is necessary to Brucella sensitive and specific. This paper aims at the preparation of Brucella polyclonal antibody, combined with immunomagnetic separation technique and quantum dot marking technology to establish a rapid detection of Brucella, and evaluate the effect of the detection method. The content of this research include the following two aspects: preparation and identification of Brucella using 1. rabbit polyclonal antibody of Brucella 16M vaccine, immunization of New Zealand white rabbits, preparation The polyclonal antibody, the antibody was purified by saturated ammonium sulfate salting out method. The establishment of indirect ELISA method, using matrix method to optimize the reaction conditions, including antigen concentration, serum concentration, coating condition, blocking solution, blocking time, the antigen antibody reaction time, concentration of two two anti, anti reaction time and time of color, determination of antibody titer and specificity and by indirect ELISA optimization method; Determination of purity of polyclonal antibody by SDS-PAGE method; Determination of protein assay kit using polyclonal antibody against Bradford protein concentration. The results showed that the optimal conditions of indirect ELISA method for antigen: the best conditions for the 37 C was 1 h, the concentration of the sealed liquid for 1:40, 5% skim milk powder, the best blocking time is 1 h, the concentration of serum 1:16000 for the best work, the best antigen antibody reaction time is 1.5 h, the best anti two The concentration of 1:10000, two anti reaction time is 0.5 h, the best color time for 10 min; 1, the antibody titer was 1:256000,2, antibody titer of 1:2048000 antibody; specificity, O157 and Escherichia coli, Listeria bacteria Lester, Proteus mirabilis, Enterobacter cloacae, Enterobacter sakazakii, Klebsiella pneumoniae Bao Shizhi coli, Shigella, Staphylococcus aureus, Serratia marcescens, Salmonella antibody cross reactivity; high purity; No. 1 and No. 2 antibody protein content respectively for the establishment and evaluation of 14.091 mg/m L and 17.454 mg/m L.2. detection method to the carboxylic beads and preparation of Brucella polyclonal antibody covalently coupling, preparation of magnetic nano probes for identification of Brucella; carboxylated Brucella egg yolk antibody pre preparation of fluorescent quantum dots and laboratory (Ig Y) covalent coupling, preparation of quantum dot fluorescent probe identification of brucella, These two kinds of biological probes to establish a new method based on the detection of Brucella, magnetic nano probe - Quantum Dots Fluorescent Probe of Brucella sandwich complexes, fluorescence detection of complexes by fluorescence spectrometer, a linear fluorescence intensity and the concentration of the bacteria liquid regression equation, thus realizing the Brucella fast. Quantitative detection. In the process of establishing a detection method, the magnetic nano probe dosage, preconcentration time, dosage of quantum dots and mark time optimization, sensitivity evaluation method, specificity and reproducibility, and the method of artificial meat and milk samples were detected. The results showed the best conditions, magnetic nano bio probe preparation for 100 L and 122.178 G using rabbit polyclonal antibody of 37 DEG C to 2 h coupling; quantum optimal point biological probe preparation Brucella detection method of the optimum reaction conditions for 10 L QDs and 262 g Ig 2 h. Y to 37 DEG C coupling established: magnetic nano probes the best dosage is 100 L, the best enrichment time is 45 min, QD probes the best dosage is 500 L, the best time is 60 min marker; detection this method is about 105 min, there was a linear relationship between the logarithm of the fluorescence intensity and the concentration of bacterium of reaction product, linear equation y=21.763x+78.371 (R2=0.9983), the detection limit is 103 CFU/m L; specific experimental results show that the method can identify the sheep and swine Brucella, but not the identification of Escherichia coli O157 and Listeria by Lester bacteria, better specificity; in milk samples and samples of simulation simulation gravy detection limit was 102 CFU/m L, to improve the detection effect in simulated samples. In summary, the preparation of Brucella polyclonal antibody, which can be identified The magnetic nano bio probe and quantum dot fluorescent probe not of Brucella, and establish a method for rapid detection of magnetic separation technique and quantum dot labeling technique based on the detection of Brucella. The detection limit is low, and has good specificity, good stability, can also be a good application in the detection of simulated samples in this work, a rapid detection method with our own intellectual property rights. Brucella

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R155

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