【作者】 陶晓奇；

【导师】 沈建忠； 江海洋；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2014， 博士

【摘要】 食品中兽药等化合物残留是影响世界各国食品安全的主要因素,不断发展高效、广谱、高灵敏的残留检测技术是保障食品安全的有效途径。酰胺醇类药物因其广谱抗菌特点在兽医临床上使用非常广泛,但该类药物经食物链进入人体对人类健康构成威胁,其中氯霉素已在全球禁用于食品动物。本论文开展动物性食品中酰胺醇类残留的高灵敏化学发光免疫检测方法的研究,旨在为酰胺醇类残留监控提供快速、灵敏的检测手段。本论文建立了检测牛奶和鸡肉中氯霉素的间接竞争化学发光免疫分析方法,检测限分别为0.00092μg·kg-1和0.0010μg·kg-1。在0.005-0.1μg·kg-1三个浓度添加时,回收率为87.6%-118.8%,变异系数均小于15%；建立了检测猪肉中氟苯尼考和甲砜霉素的间接竞争化学发光免疫分析方法,检测限分别为0.015μg·kg-1和0.03μg·kg-1,在0.05-4.0μg·kg-1(氟苯尼考)或0.1-9.0μg·kg-1(甲砜霉素)三个浓度添加时,回收率均在83.0%-102.2%之间,变异系数小于15%；建立了检测鸡肉中氟苯尼考和氟苯尼考胺的间接竞争化学发光免疫分析方法,样本稀释10倍时,检测限分别为0.17μg·kg-1(氟苯尼考)和0.12μg·kg-1(氟苯尼考胺),样本稀释100倍时,检测限分别为1.63μg·kg-1(氟苯尼考)和1.18μg·kg-1(氟苯尼考胺)。以0.5-300μg·kg-1六个浓度添加,回收率在70.3%-102.0%范围内,变异系数小于15%。上述方法的灵敏度、检测限、回收率以及稳定性等指标均满足我国和欧盟兽药残留分析的要求,与传统ELISA方法相比,灵敏度提高了10倍左右。本论文研究建立了基于单酶和复酶标记的同时检测牛奶中氯霉素(酰胺醇类)和克伦特罗(β-兴奋剂类)残留的混合化学发光免疫分析方法。在辣根过氧化物酶(HRP)标记的反应体系中,氯霉素和克伦特罗的检测限分别为0.006μg·L-1和0.02μg·L-1。在HRP和碱性磷酸酶(ALP)标记的反应体系中,氯霉素和克伦特罗的检测限分别为0.008μg·L-1和0.023μg L-1。在氯霉素(0.01-0.3μg·L-1)和克伦特罗(0.05-1.0μg·L-1)单独以及共同添加中,回收率分别为80.0%—90.0%(HRP体系)和86.1%-96.7%(HRP和ALP体系),变异系数小于15%,满足我国和欧盟兽药残留分析的要求。本论文探索了基于磁珠分离和纳米金标记技术检测牛奶中氯霉素残留的新型化学发光免疫分析方法,与相同单克隆抗体建立的化学发光免疫分析方法相比,灵敏度提高了近30倍。合成了纳米金颗粒与酶标氯霉素单克隆抗体的偶联物,氯霉素抗原与磁珠的共价结合物,在此基础上建立了检测牛奶中氯霉素的直接竞争化学发光免疫分析方法。灵敏度IC50为0.017μg·L-1(乙酸乙酯提取)和0.17μg·L-1(直接稀释)。以0.01-0.3μg L-1三个浓度(乙酸乙酯提取)和0.1-3.0μg.L-1三个浓度添加时(直接稀释),回收率为80.0%-106.6%,且变异系数小于15%,符合我国和欧盟兽药残留分析的要求。更多还原

【Abstract】 Residues of veterinary drugs in foods are the main factors affecting the global food safety, Development of efficient, reliable and high sensitive detection techniques in veterinary drug residues is an effective way to ensure food safety. Amphenicols were widely used in veterinary practices for the prevention and treatment of many bacterial infections, due to the broad-spectrum antibacterial characteristics. However, the residues of these drugs occuring in various animal origin products present a potential danger to human health, and chloramphenicol (CAP) was now forbidden in food producing animals in the world. This work aims to establish more sensitive chemiluminescent immunoassays to screen amphenicol residues in animal-derived food, and then provide rapid and sensitive analytical methods of monitoring amphenicol residues.Firstly, a competitive indirect chemiluminescent enzyme-liked immunoassay (CL-ciELISA) for chloramphenicol (CAP) residues in milk and chicken muscle has been developed, with a detection limit (LOD) of0.00092μg·kg-1(milk) and0.0010μg·kg-1(chicken muscle). When CAP was spiked in milk and chicken muscle at levels of0.05-0.1μg·kg-1, recoveries ranged from87.6%-118.8%, with coefficients of variation (CVs)<15%, respectively. Then, a CL-ciELISA for FF and TAP residues in pork has been developed, with LOD of0.015μg·kg-1for FF and LOD of0.015μg·kg-1for TAP. When FF and TAP was spiked in pork at levels of0.05-4.0μg·kg-1or0.1-9.0μg·kg-1, respectively, recoveries ranged from83.0%-102.2%, with CVs<15%. Finally, a CL-ciELISA for FF and FFA residues in chicken muscle has been developed, with LODs of0.17μg·kg-1(10-fold dilution) and1.63μg·kg-1(100-fold dilution) for FF,0.12μg·kg-1(10-fold dilution) and1.18μg·kg-1(100-fold dilution) for FFA. When FF and FFA were spiked in chicken muscle at levels of0.5-300μg·kg-1, respectively, recoveries ranged from70.3%-102.0%, and CVs<15%. The sensitivity, LOD, recovery and robustness of the three above CL-ELSIAs for amphenicols meet Chinese and EU’s premises in detection methods of veterinary drug reidues. Compared with ELISA methods, the CL-ELISAs can improve the sensitivity about10times.Two novel hybrid chemiluminescent immunoassays using HRP-luminol chemiluminescence system and two different enzymatic systems—HRP and alkaline phosphatase (ALP) chemiluminescent systems were established and sucessfully applied to simultaneous detection of chloramphenicol (CAP) and clenbuterol (CLE) in milk, with LODs of0.006μg·kg-1for CAP and0.02μg·kg-1for CLE (HRP system),0.008μg·kg-1for CAP and0.023μg·kg-1for CLE (HRP and ALP system), respectively. When CAP (0.01-0.3μg·L-1) and CLE (0.05-1.0μg·L-1) were individually or simultaneously fortified in milk, the recoveries were found to be between80.0%and96.7%, with CVs<15%, meeting Chinese and EU’s premises in detection methods of veterinary drug reidues.In this study, a competitive direct chemiluminescent immunoassay based on a magnetic beads (MBs) separation and gold nanoparticles (AuNPs) labeling technique to detect CAP in milk was explored and can improve the sensitivity about30times, compared with CL-ELISA method based on the same monoclonal antibody. Horseradish peroxidase (HRP)-labelled anti-CAP monoclonal antibody conjugated with AuNPs and antigen-immobilized MBs were prepared. Then, a competitive direct chemiluminescent immunoassay for CAP in milk was established. The IC50values of chemiluminescence magnetic nanoparticles immunoassay (CL-MBs-Nano-Immunoassay) were0.017μg·L-1for extract method I (ethyl acetate extraction) and0.17μg·L-1for extract method II (direct dilutions). When CAP was spiked in milk at levels of0.01-0.3μg·L-1(ethyl acetate extraction) and0.1-3.0μg·L-1(direct dilutions), recoveries were in the range of80.0%-106.6%, and CVs were all<15%, meeting meet Chinese and EU’s premises in detection methods of veterinary drug reidues.更多还原