单增李斯特菌荧光分子印迹材料合成与检测方法的建立

发布时间：2018-11-04 08:43

【摘要】：单增李斯特菌是一种广泛的食源性致病菌,它可以通过奶制品、肉类等引起细菌性食物中毒,是食品安全领域的重点关注对象。而传统的单增李斯特菌的检测方法虽然相对准确,但检测周期较长且步骤繁琐,因此,开发一种灵敏、快速、简单、低成本的检测单增李斯特菌的方法已经成为现今食品卫生行业的重要环节。近年来,量子点作为一种新型的荧光探针,具有良好的光稳定性,本论文以巯基乙酸为稳定剂,一步法合成水溶性CdTe量子点,再在壳聚糖上修饰丙烯酰基的侧链合成NAC功能单体,然后在EDC的作用下将其与CdTe量子点进行连接形成NAC-QDs复合物,再将NAC-QDs作为功能单体,以单增李斯特菌为模板分子,TRIM和DVB为交联剂,过氧化苯甲酰引发聚合反应,采用基于Pickering乳液与分子印迹技术相结合,制备了对单增李斯特菌特异性识别的荧光分子印迹聚合物。本实验对聚合反应的条件进行优化和分析,对分子印迹聚合物的吸附性能和选择性识别的能力进行测定。利用扫描电镜、荧光分光光度计、傅立叶红外光谱、倒置荧光显微镜对荧光分子印迹聚合物进行表征,并对吸附性能实验的数据进行分析,结果表明:在最优的合成条件下,该聚合物通过印迹孔穴特异性识别单增李斯特菌,具有较好的选择性,吸附2 h后基本达到吸附平衡,吸附容量约为1644.45 CFU/mg。本文建立了一种基于荧光分子印迹材料对单增李斯特菌的检测方法,经荧光可视和直接计数,两步判断结果。随着单增李斯特菌菌悬液浓度的增大,荧光分子印迹聚合物的猝灭效果更明显,荧光可视检测法的最低检出限为10~3CFU/mL:直接计数法最低检出量的菌浓约为75 CFU/mL。为了验证实验建立检测方法的实用性,对人工污染的牛奶和猪肉中的单增李斯特菌进行检测分析,在牛奶和猪肉中依次加入不同浓度的单增李斯特菌液,经检测牛奶的荧光可视法的最低检出限为1.6×103CFU/g,猪肉的荧光可视法的最低检出限为6.5×10~2CFU/g;直接计数牛奶中最低检出量的菌浓约为120CFU/g,猪肉中最低检出量的菌浓约为76CFU/g。
[Abstract]:Listeria monocytogenes (Listeria monocytogenes) is a kind of foodborne pathogenic bacteria which can cause bacterial food poisoning through dairy products meat etc. It is the focus of attention in the field of food safety. Although the traditional method for detection of Listeria monocytogenes is relatively accurate, the detection period is long and the steps are tedious. Therefore, a sensitive, rapid and simple method is developed. Low-cost detection of Listeria monocytogenes has become an important part of the food hygiene industry. In recent years, as a new fluorescent probe, quantum dots have good photostability. In this paper, water-soluble CdTe quantum dots were synthesized by one-step method using mercaptoacetic acid as stabilizer. NAC functional monomer was synthesized by modifying the side chain of acrylyl group on chitosan, and then connected with CdTe quantum dot under the action of EDC to form NAC-QDs complex. NAC-QDs was used as functional monomer and Listeria monocytogenes as template molecule. Fluorescence molecularly imprinted polymers (FMIPs) specifically recognized by Listeria monocytogenes were prepared by using TRIM and DVB as crosslinkers and benzoyl peroxide initiated polymerization by combining Pickering emulsion with molecular imprinting technique. The conditions of polymerization were optimized and analyzed, and the adsorption properties and selective recognition ability of molecularly imprinted polymers were determined. Fluorescence molecularly imprinted polymers were characterized by scanning electron microscope, fluorescence spectrophotometer, Fourier transform infrared spectroscopy and inverted fluorescence microscope. The polymer can specifically identify Listeria monocytogenes by imprinting holes, and has good selectivity. After 2 h adsorption, the adsorption equilibrium is basically achieved, and the adsorption capacity is about 1644.45 CFU/mg.. In this paper, a method for the detection of Listeria monocytogenes based on fluorescent molecular imprinting material was established. The results were judged by fluorescence visible and direct counting. With the increase of the suspension concentration of Listeria monocytogenes, the quenching effect of fluorescent molecularly imprinted polymer was more obvious. The detection limit of fluorescence visual detection method was 10 ~ 3 CFU / mL, and the concentration of bacteria detected by direct counting method was about 75 CFU/mL.. In order to verify the practicability of the method established in the experiment, Listeria monocytogenes in artificially contaminated milk and pork were detected and analyzed. Different concentrations of Listeria monocytogenes were added to milk and pork in turn. The detection limit of fluorescence visual method was 1.6 脳 10 ~ 3 CFU / g, and that of pork was 6.5 脳 10 ~ (-2) CFU / g. The concentration of bacteria detected in milk directly was about 120 CFU / g, and that in pork was about 76 CFU / g.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS207.4;O657.3

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