农产品中四种常见真菌毒素免疫检测技术研究
发布时间：2019-05-22 02:49
【摘要】：真菌毒素(Mycotoxin)是一类由真菌产生的小分子次级代谢产物,农作物在生长、收获、贮存和运输等环节均易受到真菌毒素污染,现已发现400多种真菌毒素,大部分具有致癌、致畸和致突变作用且混合污染时毒性会显著增强。真菌毒素可通过污染谷物和饲料或经动物源性食品进入食物链,对人类和动物健康造成严重伤害,近年来已成为食品安全领域的研究重点。真菌毒素现有检测方法包括生物鉴定法、化学分析法、仪器分析法和免疫分析法等。生物鉴定法专一性不强；化学分析法精确度低；仪器分析灵敏度高,可准确定量,但样本前处理复杂并且检测仪器昂贵,难以在基层实验室推广使用。免疫分析方法快速、灵敏度高、特异性好,并且成本较低,近年来在真菌毒素检测领域发展迅速。本课题选取农产品中常见且危害较大的黄曲霉毒素B1 (Alfatoxin B1, AFB1)、赭曲霉毒素A (Ochratoxin A, OTA)、伏马毒素B1 (Fumonisin B1, FB1)和玉米赤霉烯酮(Zeralenone, ZEN)为检测对象,以单克隆抗体作为生物识别元件,基于竞争ELISA和免疫层析原理,结合信号放大、免疫传感、抗体芯片等技术,建立多种真菌毒素的快速、敏感、高通量免疫检测方法。本论文的主要研究内容和结果如下：1.赭曲霉毒素A的间接竞争ELISA定量检测技术对OTA单克隆抗体2D8的免疫学性质进行鉴定,以此抗体为基础建立间接竞争酶联免疫检测法(ci-ELISA)。抗体2D8效价为1：1.024×106,亲和常数Ka=3.75×109L/M,重链为IgG1型,与赭曲霉毒素B (Ochratoxin B, OTB)的交叉反应率为5.4%,与AFB1、FB1、ZEN、桔青霉毒素(Citrinin, CIT)、展青霉毒素(Patulin, PAT)和呕吐毒素(Deoxynivalenol, DON)均无交叉反应。建立的ci-ELISA检测限为0.08 ng/mL,检测范围0.12-1.18 ng/mL,加标回收率可达91.2-110.3%,与商品化ELISA试剂盒相关性较好,可用于谷物及饲料样本中OTA的定量检测与分析,也为后续其它免疫检测方法的建立奠定基础。2.基于免疫磁珠和生物素-链霉亲和素的间接竞争ELISA定量检测玉米赤霉烯酮为缩短反应时间,提高检测灵敏度,以纳米磁珠作为固相载体包被ZEN单克隆抗体,基于竞争反应原理,利用生物素-链霉亲和素系统,建立新型酶联免疫检测方法(MNP-bsELISA).该方法检测限为0.04 ng/mL,检测范围0.07-2.41ng/mL,加标回收率为92.8-111.9%,变异系数小于10%,对天然样本中ZEN的定量结果与LC-MS/MS方法具有良好的相关性。3.赭曲霉毒素A磁珠-电化学免疫传感检测技术优化以磁珠为固相载体的免疫竞争反应,选取最佳模式并结合电化学信号检测技术,建立了OTA电化学免疫传感检测技术。该方法的检测下限达到0.007ng/mL,检测范围为0.01-0.82 ng/mL,加标回收率为78.7-91.6%,标准偏差和变异系数均小于15%,稳定性较好,可实现样本中OTA的痕量检测。4.赭曲霉毒素A和玉米赤霉烯酮二联免疫层析试纸条研制基于免疫层析原理,分别建立了二联定性和定量胶体金免疫层析检测方法用于谷物和饲料中OTA和ZEN的检测。其中二联定性胶体金免疫层析试纸条对OTA和ZEN的检测限分别为0.625 ng/mL和1.25 ng/mL,二联定量胶体金免疫层析试纸条对OTA的检测限为0.25 ng/mL,检测区间为0.32-12.16 ng/mL;对ZEN的检测限为0.5 ng/mL,检测区间为0.58-39.72 ng/mL;两种真菌毒素在玉米、小麦和饲料样品中加标回收率为79.6-105.2%。制备的二联免疫层析试纸条对样本的检测结果与LC-MS/MS相关性较好,可分别满足样本中OTA和ZEN的快速定性检测与定量分析。5.同时检测四种真菌毒素的高通量抗体芯片定量技术真菌毒素混合污染情况较为常见且毒性会显著增强,为对样本中四种常见真菌毒素(AFB1、OTA、FB1和ZEN)进行高通量、同时定量检测,以硝酸纤维素膜为基板,利用生物素.链霉亲和素系统,建立抗体芯片检测模式。该方法对AFB1、OTA、FB1、ZEN的检测限分别为0.21、0.19、0.24和0.09 ng/mL,检测区间分别为0.47-55.69、0.48-127.11、0.56-92.57和0.22-31.36 ng/mL。在实际样本中的回收率为79.2-113.4%,变异系数小于10%,稳定性较好,可对真菌毒素进行多重定量检测,在真菌毒素混合污染监测领域具有广阔的应用前景。综上所述,本研究将真菌毒素特异性单克隆抗体与磁性纳米材料、生物素链霉亲和素、电化学、芯片等信号放大和高通量检测体系相结合,分别建立了ci-ELISA、MNP-bsELISA、磁珠电化学免疫传感器、二联定性和定量胶体金免疫层析试纸条和抗体芯片多重检测技术,可满足农产品中真菌毒素快速便捷、高灵敏和高通量的检测需求,为农产品、食品和饲料中真菌毒素污染监测体系的建立提供技术支撑,也为其他小分子分析物的检测提供参考与策略。
[Abstract]:Mycotoxin is a kind of small-molecule secondary metabolite produced by the fungus, and the crops are easy to be polluted by the mycotoxin in the aspects of growth, harvesting, storage and transportation, and more than 400 mycotoxins have been found, most of which are carcinogenic, The toxicity of teratogenic and mutagenic effects and mixed contamination is significantly enhanced. The mycotoxin can enter the food chain through the pollution of grain and feed or animal-derived food, and has serious harm to human and animal health, and has become the research focus in the field of food safety in recent years. The existing detection methods of mycotoxins include the biological identification method, the chemical analysis method, the instrument analysis method and the immunoassay method, and the like. The biological identification method is not strong in specificity, the chemical analysis method is low in precision, the analytical sensitivity of the instrument is high, the accuracy can be accurately quantified, the pretreatment of the sample is complicated and the detection instrument is expensive, and the use is difficult to be popularized in the basic-layer laboratory. The immune analysis method is rapid, high in sensitivity, good in specificity and low in cost, and has developed rapidly in the field of mycotoxin detection in recent years. Aflatoxin B1 (AFB1), aspergillotoxin A (OTA), fumonisin B1 (FB1) and Zealenone (ZEN), which are common and harmful to the agricultural products, are selected as the detection target, and the monoclonal antibody is used as the biological identification element. Based on the principle of competitive ELISA and immunochromatography, the rapid, sensitive and high-throughput immunodetection method of various mycotoxins is established by combining the techniques of signal amplification, immunosensing, antibody chip and the like. The main contents and results of this thesis are as follows:1. The immunological properties of the OTA monoclonal antibody 2D8 were identified by indirect competitive ELISA. The indirect competitive enzyme-linked immunosorbent assay (CI-ELISA) was established based on the antibody. The antibody 2D8 has a potency of 1: 1.024-106, a affinity constant Ka = 3.75-109L/ M, a heavy chain of an IgG1 type, a cross-reaction rate of 5.4% with an aspergiloxin B (Ochratoxin B, OTB), and AFB1, FB1, ZEN, Penicillium citrinin (CIT), Paecilomyces (Patulin, PAT), and a vomiting toxin (Deoxynivalenol, DON) No cross-reaction. The detection limit of ci-ELISA is 0.08 ng/ mL, the detection range is 0.12-1.18 ng/ mL, the recovery rate of the spike can reach 91.2-110.3%, the correlation with the commercial ELISA kit is good, the quantitative detection and analysis of the OTA in the grain and the feed sample can be used, and the foundation for the establishment of the subsequent other immunodetection methods is also provided. the method is based on the indirect competitive ELISA of the immunomagnetic beads and the biotin-streptavidin to quantitatively detect the corn trichoderma ketene to shorten the reaction time, improve the detection sensitivity, coat the ZEN monoclonal antibody as a solid phase carrier by using the nano magnetic beads as a solid phase carrier, and based on the principle of the competition reaction, A new enzyme-linked immunosorbent assay (MNP-bsELISA) was established using a biotin-streptavidin system. The detection limit of the method is 0.04 ng/ mL, the detection range is 0.07-2.41 ng/ mL, the recovery of the spike is 92.8-111.9%, the coefficient of variation is less than 10%, and the quantitative result of ZEN in the natural sample has a good correlation with the LC-MS/ MS method. The immune competitive reaction of the magnetic bead as the solid-phase carrier is optimized by using the magnetic bead-electrochemical immunosensing detection technology of the aspergillosis toxin A. The best mode is selected and the electrochemical signal detection technology is combined to establish the OTA electrochemical immunosensing detection technology. The detection limit of the method is 0.007 ng/ mL, the detection range is 0.01-0.82 ng/ mL, the recovery rate of the spike is 78.7-91.6%, the standard deviation and the coefficient of variation are all less than 15%, the stability is good, and the trace detection of the OTA in the sample can be realized. Based on the principle of immunochromatography, an immunochromatographic method for the detection of OTA and ZEN in grain and feed was established based on the principle of immunochromatography. The detection limit for OTA and ZEN is 0.625 ng/ mL and 1.25 ng/ mL respectively. The detection limit of the two-linked qualitative colloidal gold immunochromatographic test strip to OTA is 0.25 ng/ mL, the detection interval is 0.32-12.16 ng/ mL, the detection limit to ZEN is 0.5 ng/ mL, and the detection interval is 0.58-39.72 ng/ mL; The recovery of two mycotoxins in corn, wheat and feed samples was 79.6-105.2%. The detection results of the prepared two-linked immunochromatographic test strip and the LC-MS/ MS are better than that of the LC-MS/ MS, and the rapid qualitative detection and quantitative analysis of OTA and ZEN in the samples can be respectively met. the high-flux antibody chip quantitative technical mycotoxin mixed pollution condition of the four mycotoxins is detected at the same time, and the toxicity is obviously enhanced, and the high-flux antibody chip quantitative detection is carried out on the four common mycotoxins (AFB1, OTA, FB1 and ZEN) in the sample, Taking the nitrocellulose membrane as a substrate, and using biotin. And the streptavidin system is used for establishing an antibody chip detection mode. The detection limits of AFB1, OTA, FB1 and ZEN were 0.21, 0.19, 0.24 and 0.09 ng/ mL, respectively. The detection interval was 0.47-55.69, 0.48-127.11, 0.56-92.57 and 0.22-31.36 ng/ mL, respectively. The recovery rate of the actual sample is 79.2-113.4%, the coefficient of variation is less than 10%, the stability is good, and the mycotoxin can be tested for multiple quantitative detection, and has wide application prospect in the field of the mixed pollution monitoring of the mycotoxin. in conclusion, that present study combine the specific monoclonal antibody of the mycotoxin with the signal amplification and high-flux detection system of the magnetic nano-material, the biotin-streptavidin, the electrochemistry, the chip and the like, and respectively establish the ci-ELISA, the MNP-bsELISA and the magnetic bead electrochemical immunosensor, the two-way qualitative and quantitative colloidal gold immunochromatographic test strip and the antibody chip multiple detection technology can meet the rapid and convenient, high-sensitivity and high-flux detection requirement of the mycotoxin in the agricultural product, and provides the technical support for the establishment of the mycotoxin pollution monitoring system in the agricultural products, the food and the feed, And also provides reference and strategy for the detection of other small molecular analytes.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S133
[Abstract]:Mycotoxin is a kind of small-molecule secondary metabolite produced by the fungus, and the crops are easy to be polluted by the mycotoxin in the aspects of growth, harvesting, storage and transportation, and more than 400 mycotoxins have been found, most of which are carcinogenic, The toxicity of teratogenic and mutagenic effects and mixed contamination is significantly enhanced. The mycotoxin can enter the food chain through the pollution of grain and feed or animal-derived food, and has serious harm to human and animal health, and has become the research focus in the field of food safety in recent years. The existing detection methods of mycotoxins include the biological identification method, the chemical analysis method, the instrument analysis method and the immunoassay method, and the like. The biological identification method is not strong in specificity, the chemical analysis method is low in precision, the analytical sensitivity of the instrument is high, the accuracy can be accurately quantified, the pretreatment of the sample is complicated and the detection instrument is expensive, and the use is difficult to be popularized in the basic-layer laboratory. The immune analysis method is rapid, high in sensitivity, good in specificity and low in cost, and has developed rapidly in the field of mycotoxin detection in recent years. Aflatoxin B1 (AFB1), aspergillotoxin A (OTA), fumonisin B1 (FB1) and Zealenone (ZEN), which are common and harmful to the agricultural products, are selected as the detection target, and the monoclonal antibody is used as the biological identification element. Based on the principle of competitive ELISA and immunochromatography, the rapid, sensitive and high-throughput immunodetection method of various mycotoxins is established by combining the techniques of signal amplification, immunosensing, antibody chip and the like. The main contents and results of this thesis are as follows:1. The immunological properties of the OTA monoclonal antibody 2D8 were identified by indirect competitive ELISA. The indirect competitive enzyme-linked immunosorbent assay (CI-ELISA) was established based on the antibody. The antibody 2D8 has a potency of 1: 1.024-106, a affinity constant Ka = 3.75-109L/ M, a heavy chain of an IgG1 type, a cross-reaction rate of 5.4% with an aspergiloxin B (Ochratoxin B, OTB), and AFB1, FB1, ZEN, Penicillium citrinin (CIT), Paecilomyces (Patulin, PAT), and a vomiting toxin (Deoxynivalenol, DON) No cross-reaction. The detection limit of ci-ELISA is 0.08 ng/ mL, the detection range is 0.12-1.18 ng/ mL, the recovery rate of the spike can reach 91.2-110.3%, the correlation with the commercial ELISA kit is good, the quantitative detection and analysis of the OTA in the grain and the feed sample can be used, and the foundation for the establishment of the subsequent other immunodetection methods is also provided. the method is based on the indirect competitive ELISA of the immunomagnetic beads and the biotin-streptavidin to quantitatively detect the corn trichoderma ketene to shorten the reaction time, improve the detection sensitivity, coat the ZEN monoclonal antibody as a solid phase carrier by using the nano magnetic beads as a solid phase carrier, and based on the principle of the competition reaction, A new enzyme-linked immunosorbent assay (MNP-bsELISA) was established using a biotin-streptavidin system. The detection limit of the method is 0.04 ng/ mL, the detection range is 0.07-2.41 ng/ mL, the recovery of the spike is 92.8-111.9%, the coefficient of variation is less than 10%, and the quantitative result of ZEN in the natural sample has a good correlation with the LC-MS/ MS method. The immune competitive reaction of the magnetic bead as the solid-phase carrier is optimized by using the magnetic bead-electrochemical immunosensing detection technology of the aspergillosis toxin A. The best mode is selected and the electrochemical signal detection technology is combined to establish the OTA electrochemical immunosensing detection technology. The detection limit of the method is 0.007 ng/ mL, the detection range is 0.01-0.82 ng/ mL, the recovery rate of the spike is 78.7-91.6%, the standard deviation and the coefficient of variation are all less than 15%, the stability is good, and the trace detection of the OTA in the sample can be realized. Based on the principle of immunochromatography, an immunochromatographic method for the detection of OTA and ZEN in grain and feed was established based on the principle of immunochromatography. The detection limit for OTA and ZEN is 0.625 ng/ mL and 1.25 ng/ mL respectively. The detection limit of the two-linked qualitative colloidal gold immunochromatographic test strip to OTA is 0.25 ng/ mL, the detection interval is 0.32-12.16 ng/ mL, the detection limit to ZEN is 0.5 ng/ mL, and the detection interval is 0.58-39.72 ng/ mL; The recovery of two mycotoxins in corn, wheat and feed samples was 79.6-105.2%. The detection results of the prepared two-linked immunochromatographic test strip and the LC-MS/ MS are better than that of the LC-MS/ MS, and the rapid qualitative detection and quantitative analysis of OTA and ZEN in the samples can be respectively met. the high-flux antibody chip quantitative technical mycotoxin mixed pollution condition of the four mycotoxins is detected at the same time, and the toxicity is obviously enhanced, and the high-flux antibody chip quantitative detection is carried out on the four common mycotoxins (AFB1, OTA, FB1 and ZEN) in the sample, Taking the nitrocellulose membrane as a substrate, and using biotin. And the streptavidin system is used for establishing an antibody chip detection mode. The detection limits of AFB1, OTA, FB1 and ZEN were 0.21, 0.19, 0.24 and 0.09 ng/ mL, respectively. The detection interval was 0.47-55.69, 0.48-127.11, 0.56-92.57 and 0.22-31.36 ng/ mL, respectively. The recovery rate of the actual sample is 79.2-113.4%, the coefficient of variation is less than 10%, the stability is good, and the mycotoxin can be tested for multiple quantitative detection, and has wide application prospect in the field of the mixed pollution monitoring of the mycotoxin. in conclusion, that present study combine the specific monoclonal antibody of the mycotoxin with the signal amplification and high-flux detection system of the magnetic nano-material, the biotin-streptavidin, the electrochemistry, the chip and the like, and respectively establish the ci-ELISA, the MNP-bsELISA and the magnetic bead electrochemical immunosensor, the two-way qualitative and quantitative colloidal gold immunochromatographic test strip and the antibody chip multiple detection technology can meet the rapid and convenient, high-sensitivity and high-flux detection requirement of the mycotoxin in the agricultural product, and provides the technical support for the establishment of the mycotoxin pollution monitoring system in the agricultural products, the food and the feed, And also provides reference and strategy for the detection of other small molecular analytes.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S133
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