应用上转发光免疫层析技术快速定量检测食品中黄曲霉毒素（M1、B1）的方法建立

发布时间：2018-09-11 21:24

【摘要】：目的：以上转发光(Up-Converting Phospher, UCP)材料作为标记物,与经典免疫层析方法结合,建立可对奶制品中黄曲霉毒素M1及粮食中黄曲霉毒素B1直接进行现场检测的上转发光免疫层析(up-converting phosphor technology-based lateral-flow, UPT-LF)'决速定量检测方法。方法：1.上转发光免疫层析试纸制备：分别以黄曲霉毒素M1(AFM1)单克隆抗体,黄曲霉毒素Ml (AFM1)与小牛血清白蛋白(BSA)偶联物：AFM1-BSA;黄曲霉毒素Bl (AFB1)单克隆抗体,黄曲霉毒素B1(AFB1)与小牛血清白蛋白(BSA)偶联物：AFB1-BSA作为特异性生物探针,以UCP颗粒作为标记物,采用竞争免疫反应作为检测模型,分别建立免疫层析试纸：黄曲霉毒素Ml上转发光免疫层析检测试纸(UPT-LF-AFM1)及黄曲霉毒素B1上转发光免疫层析检测试纸(UPT-LF-AFB1).2.上转发光免疫层析检测试纸性能评价：1) UPT-LF-AFM1:配置系列浓度AFM1毒素标准品,评价UPT-LF-AFM1包括检测敏感性、线性、精密性在内的检测性能。对实际牛奶及奶粉样品同时进行UPT-LF-AFM1及LC-MS检测,评价UPT-LF-AFM1对实际样品的定性及定量检测能力。2)UPT-LF-AFB1:配置系列浓度AFB1毒素标准品,评价UPT-LF-AFB1包括检测敏感性、线性、精密性在内的检测性能,同时以其余相关生物毒素标准品评价UPT-LF-AFB1检测特异性。将AFB1毒素标准品添加到实际粮食样品中作为检测样品,评价UPT-LF-AFB1对实际样品的检测能力及其耐受性。结果：1.上转发光免疫层析试纸制备：1) UPT-LF-AFM1:将抗AFM1单克隆抗体、羊IgG分别与UCP颗粒共价偶联,用结合物稀释液将两种结合物等比混合,以终浓度1.5mg·mL-1浸泡玻璃纤维作为结合垫。AFM1-BSA与兔抗羊IgG分别配制为0.5 mg·mL-1、0.3 mg·mL-1,以2μL·cm-1喷点于分析膜上作为检测带(T)与质控带(C)。2)UPT-LF-AFB1:将抗AFB1单克隆抗体、兔抗羊IgG分别与UCP颗粒共价偶联,用结合物稀释液将两种结合物等比混合,以终浓度1mg·mL-1浸泡玻璃纤维作为结合垫。AFB1-BSA与羊IgG均配制为0.5mg·mL-1,以2μL·cm-1喷点于分析膜上作为检测带(T)与质控带(C)。2.上转发光免疫层析检测试纸性能评价：1) UPT-LF-AFM1:对奶粉中AFM1检测敏感性可达O.lppb (μg·Kg-1)、在0.1～0.7ppb (μg·Kg-1)间具有较好线性(r=-0.99359,P=0.00641)；牛奶中AFM1检测敏感性可达0.3ppb (μg·L-1),在0.3-0.7ppb((μg·L-1)间具有较好线性(r=-0.99938,P0.05),每个浓度重复测量的变异系数小于15%。在定性检测评价中,对奶粉检测灵敏度Se=100%,特异度Sp=85%,ROC曲线下面积为0.978±0.036；牛奶检测中灵敏度Se=81%,特异度Sp=88%,ROC曲线下面积为0.891±0.099：与LC-MS结果进行x2检验,无显著差异(P0.05)。在定量检测评价中,UPT-LF-AFM1对于奶粉及牛奶中AFM1检测均具有较好回收率,与LC-MS比较无明显差异(奶粉：t=0.66,P0.05；牛奶：t=1.01,P0.05)。2) UPT-LF-AFB1:对AFB1检测敏感性可达0.03ng·mL-1,在0.03-1000ng·mL-1间具有较好线性(r=-0.98459,P0.0001),每个浓度重复检测变异系数小于10%。特异性较优,除与结构相似的AFM1存在一定程度的交叉反应外,与赭曲霉毒素、蓖麻毒素、肉毒毒素等七种毒素均无交叉反应。UPT-LF-AFB1对包括花生、大红豆、玉米等在内的15种粮食样本耐受性均较优,均可满足国标中限量标准检测要求。结论：本研究建立了可分别对奶制品中黄曲霉毒素M1及粮食中黄曲霉毒素B1进行快速定量检测的上转发光免疫层析试纸条,操作简便快捷,无需复杂前处理,具有良好的检测敏感性及特异性,对各实际样品耐受性较好,可满足食品安全中对现场快速检测的需求。
[Abstract]:OBJECTIVE: To establish an up-converting phosphor technology-based lateral-flow (UPT-LF)'determination method for aflatoxin M1 in dairy products and aflatoxin B1 in grains by using UCP materials as markers combined with classical immunochromatographic methods. Quantitative detection methods: 1. Upper-forward photoimmunochromatographic strip preparation: aflatoxin M1 (AFM1) monoclonal antibody, aflatoxin Ml (AFM1) and bovine serum albumin (BSA) conjugate: AFM1-BSA; aflatoxin Bl (AFB1) monoclonal antibody, aflatoxin B1 (AFB1) and bovine serum albumin (BSA) conjugate: AFB1 BSA as a specific biological probe, UCP particles as a marker, competitive immune response as a detection model, respectively, established immunochromatographic test paper: aflatoxin ml on the transfer of photoimmunochromatographic detection paper (UPT-LF-AFM1) and aflatoxin B1 on the transfer of photoimmunochromatographic detection paper (UPT-LF-AFB1). 2. Performance evaluation of test paper: 1) UPT-LF-AFM1: Configuration of a series of concentration of AFM1 toxin standard, evaluation of UPT-LF-AFM1 including detection sensitivity, linearity, precision, including detection performance. For actual milk and milk powder samples, UPT-LF-AFM1 and LC-MS detection were carried out simultaneously to evaluate the UPT-LF-AFM1 on the actual samples of qualitative and quantitative detection capacity.2) UPT-LF-AF 1 on the actual samples. B1: A series of AFB1 toxin standards were prepared to evaluate the detection performance of UPT-LF-AFB1, including sensitivity, linearity and precision. The specificity of UPT-LF-AFB1 was evaluated by other related biological toxin standards. RESULTS: 1. UPT-LF-AFM1: Anti-AFM1 monoclonal antibody, sheep IgG were covalently coupled with UCP granules, and the two conjugates were mixed with the conjugate diluent at the same ratio. The final concentration of 1.5mg. mL-1 immersed glass fiber was used as the binding pad. AFM1-BSA and rabbit anti-sheep IgG were matched respectively. Anti-AFB1 monoclonal antibody, rabbit anti-sheep IgG covalently coupled with UCP granules, and rabbit anti-sheep IgG covalently coupled with UCP granules. The conjugates were mixed equally with the diluent of conjugates, and the glass fibers were immersed in the final concentration of 1 mg mL-1 as a bonding pad. The results showed that: 1) UPT-LF-AFM1 had a good linearity (r=-0.99359, P=0.00641) between 0.1 and 0.7 ppb (ug.Kg-1) for the detection of AFM1 in milk powder. The sensitivity of AFM1 in milk was 0.3 ppb (ug.L-1), good linearity between 0.3 and 0.7 ppb ((ug.L-1)) (r=-0.99938, P 0.05), and the coefficient of variation of repeated measurements for each concentration was less than 15%. In qualitative evaluation, the sensitivity of the milk powder was Se=100%, specificity was Sp=85%, and the area under the ROC curve was 0.978+0.036. E = 81%, specificity Sp = 88%, ROC curve area under 0.891 + 0.099: No significant difference (P 0.05). In quantitative evaluation, UPT-LF-AFM1 has a good recovery rate for the detection of AFM1 in milk powder and milk, compared with LC-MS, there is no significant difference (milk powder: t = 0.66, P 0.05; milk: t = 1.01, P 0.05). 2) UPT-LF-AFB1: The sensitivity to AFB1 was 0.03 ng mL 1, and it had a good linearity between 0.03 and 1000 ng mL 1 (r = - 0.98459, P 0.0001). The coefficient of variation was less than 10% for each concentration. The specificity was better, and there was no cross reaction with ochratoxin, ricin, botulinum toxin and other seven toxins except for the cross reaction with similar structure of AFM1. UPT-LF-AFB1 has good tolerance to 15 kinds of grain samples, including peanut, red bean, corn and so on. All of them can meet the requirements of the national standard medium-limit detection. CONCLUSION: The UPT-LF-AFB1 can be used to detect aflatoxin M1 in dairy products and aflatoxin B1 in grain quantitatively and rapidly. It is easy to operate and needs no complex pretreatment. It has good sensitivity and specificity. It has good tolerance to various actual samples. It can meet the requirements of rapid on-site detection in food safety.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R155.5

【相似文献】