超市鲜活食品质量安全体系及有机磷农药免疫检测方法研究
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摘要
我国食品安全问题比较严重，与鲜活食品有关的安全事件时有发生，尤其是有机磷农药残留的污染，对消费者健康构成了巨大威胁。超市作为食品供应链的零售终端，正逐渐成为鲜活食品销售的主渠道。对超市鲜活食品供应链质量安全体系以及有机磷农药检测技术进行研究，不仅具有理论意义，而且在实践上可以为超市食品质量安全管理提供指导，为鲜活食品中有机磷农药检测提供快速、简单、经济的方法。
本文结合苏果超市的实证分析，在明确我国超市鲜活食品质量安全存在问题的基础上，探讨我国超市鲜活食品质量安全体系的建设以及HACCP管理体系在超市叶菜类蔬菜供应链中的应用。此外，为提高农药残留检测技术水平，本文以甲氧基有机磷农药的共有结构O，O-二甲基硫代磷酸酯为目标检测基团，自主设计并合成了三种具有不同连接臂结构的半抗原，与牛血清白蛋白偶联作为免疫原免疫新西兰大白兔，制备广谱特异性抗体。通过对不同抗体—包被原组合筛选以及试验条件的优化，建立了甲氧基有机磷农药的广谱特异性免疫检测方法，并研究了抗原异源性对抗体亲和性以及方法灵敏度的影响。获得以下主要研究结果：
(1)在调研和分析我国超市鲜活食品质量安全现状及存在问题的基础上，对我国超市鲜活食品供应链质量安全体系的建设进行了探讨，包括设置独立的鲜活食品质量安全管理机构，加大生产基地的培育、冷链设施的投入以及管理力度；制定详细的企业操作标准规范，规范供应链每一步实施的行为；建立超市内控与第三方检验相结合的检测体系：完善超市鲜活食品溯源体系；建立由政府部门、行业主管部门、媒体以及消费者对超市鲜活食品质量安全进行监督的体系，最终形成具有推广价值的超市鲜活食品供应链质量安全体系。
(2)对HACCP管理体系在超市叶菜类蔬菜供应链的应用进行了研究。确定超市叶菜类蔬菜供应链中关键控制点，对关键控制点制定关键限值，设置相关的监控程序以及纠偏措施，并提出相应的政策措施建议。
(3)以甲氧基有机磷农药的共有结构O，O-二甲基硫代磷酸酯为目标检测基团，自主设计并合成了三种具有不同连接臂结构的半抗原，经~1HNMR、~(13)CNMR以及MS鉴定，合成成功。将三种半抗原分别通过两种方法偶联于载体蛋白上，制备免疫原与包被原，经紫外光谱以及红外光谱鉴定，偶联成功。
(4)抗原异源性对抗体亲和性以及免疫方法灵敏度影响的研究结果显示：抗原异源会降低由简单连接臂抗原产生的抗体对包被原的吸附作用，降低抗体的亲和性，但是会增加由复杂连接臂抗原产生的抗体对包被原的吸附作用，增加抗体的亲和性，这可能由于偶联方法不同所引起偶联比高低不同，以及不同的反应试剂对蛋白质空间构象影响不同所造成；抗原异源性对于提高由复杂连接臂抗原产生的抗体的灵敏度具有比较重要的作用，但对于由简单连接臂抗原产生的抗体的灵敏度作用则相反。具有简单连接臂的抗原较复杂连接臂抗原更适合产生灵敏度高的抗体。
(5)通过对免疫原免疫新西兰大白兔获得的抗体进行筛选以及试验条件的优化，建立了甲氧基有机磷农药广谱特异性免疫检测方法。方法最佳试验条件：抗体3055稀释倍数为1：6400，包被原Ⅱ-OVA~a浓度为2μg／mL，反应介质为5％甲醇的PBS溶液，pH为6．0～7．4，溶液离子强度为0．01mol／L。方法获得的抗体亲和力以及灵敏度高，对马拉硫磷、乐果、稻丰散、亚胺硫磷、杀扑磷、杀螟硫磷、甲基对硫磷、倍硫磷等多种甲氧基有机磷农药均有较强的识别作用，其IC_(50)分别为30．1、28．9、88．3、159．7、191．7、324．0、483．9和788．9 ng／mL。应用该方法对白菜中马拉硫磷、乐果、稻丰散、亚胺硫磷以及杀扑磷的回收率进行检测，回收率位于77．1～104．7％之间，变异系数≤13．81％。
Nowadays,the food safety problems related to the fresh food have always broken out inChina.The issue of the fresh food safety,especially for organophosphorus pesticide residues,poses threat to the consumers' health.As the end of the food supply chain,the supermarketshave becoming the main channel for fresh food distribution.The food quality and safetymanagement of supermarket is at the primary stage because of the late starting.Therefore,theresearch on the quality and safety system for fresh food of supermarket and the method fordetermination of pesticides do not only have their theoretical significance,but also providessome practical advices on the quality and safety management for fresh food,and offers thequick,simple and economical method for the determination of pesticides.
The paper clarified the problems of the fresh food management of supermarket throughthe analysis of Suguo supermarket.Basing on the economic analysis of the quality and safetyfor fresh food and the investigation of the fresh food quality and safety management ofdomestic supermarkets,the construction of fresh food quality and safety system ofsupermarket in China was discussed and the application of HACCP in the leaf vegetablesupply chain of supermarket was also researched in the paper.Beside these,in order toimprove the method for the determination of pesticides,the O,O-dimethylphosphorodithioate,which is the generic structure of methoxy organophosphorus pesticide,was used as the target determinate group to generate broad specificity polycolonal antibodies.Three haptens with different spacer-arm were synthesized and conjugated to BSA to be usedas immunogens to produce the antibodies.The broad specificity immunoassay was developedthrough selecting the combinations of antibody-coating antigen and optimizing the conditionsof experiment.The influence of the antigen heterology for the affinity of antibody and thesensitivity of the method was also studied.
Through the research of this article,some beneficial conclusions have been drawn:
(1)Basing on the analysis of Suguo supermarket and the investigation of fresh foodquality and safety management of supermarket,the construction of food quality and safetysystem of supermarket in China was discussed.The food safety management organizationshould be set independently.Begin with the cultivating of the production base,the manage measures should be established separately for the three mode of fresh food supply chains,including“Production base-Supermarket”,“Production base-Merchant-Supermarket”and“Production base,Farmers' corporate organization,individual farmer-Wholesalemarket-Supermarket”.The devotion of equipments for cool chain should be increased and thepower of management should be improved.The standard of operation in every tache of thesupply chain should be done to standardize the action.The advanced method should beintroduced to develop the determination platform.The determination system should be set upby combining the supervision from supermarket and the determination from the third party.The system of tracing to the source basing on the development of technic and the databaseshould be developed.The supervision system should be constructed in every tache of thesupply chain supervised by the govemment,trade department,media and consumers.Finally,the fresh food quality and safety system of supermarket which is valuable for popularizationis formed.
(2)The application of HACCP in the leaf vegetable supply chain of supermarket wasstudied according to the implement process of HACCP.Basing on the results of hazardanalysis,the critical control points were the tache of determination before carrying away fromproduction base and the tache of acceptance by supermarket.The critical limites of pesticides,heavy metals and sense inspection,the inspection procedure and the correction measure werealso established.Some correlative policy measures were recommended.
(3)The O,O-dimethyl phosphorodithioate which is the generic structure of methoxyorganophosphorus pesticide was used as the target determinate group.Three haptens withdifferent spacer-arms were synthesized.~1HNMR,~(13)CNMR and MS spectra data supported allstructures of target molecules.The haptens were conjugated to carrier proteins forimmunogens and coating antigens by two coupling methods.UV-vis and IR spectral dataconfirmed the structures of the final conjugates.
(4)The effect of antigen heterology on antibody affinity and immunoassay sensitivitywas also studied.The results showed as follows:The titer difference between homologousand heterologous was great.The heterology in spacer arm may be not available for theantigen recognition by antibodies generated from immunogen with simple spacer arm.Thetiter difference may be due to the different coupling methods resulting in different haptenloads and different influence in the space structure of protein.Antigen heterology is moreimportant for the antiserum immunized by the hapten with complicated spacer arm than thesimple spacer arm to improve the sensitivity of immunoassay.The hapten with simple spacerarm may be more conformable to produce the antibody with high sensitivity.
(5)A general and broad class-specific enzyme-linked immunosorbent assay was developed for the O,O-dimethyl organophosphorus pesticides.The optimized conditions ofassay used antibody 3055 at a dilution of 1:6400,coating antigenⅡ-OVA~a at 2μg/mL,andanalyte in 5% methanol-0.01 mol/L PBS buffer,pH is between 6.0～7.4.The IC50 values ofthe assay,under optimized conditions,were estimated to be 30.1 ng/mL for malathion,28.9ng/mL for dimethoate,88.3 ng/mL for phenthoate,159.7 ng/mL for phosmet,191.7 ng/mL formethidathion,324.0 ng/mL for fenitrothion,483.9 ng/mL for methyl parathion,and 788.9ng/mL for fenthion.Recoveries of malathion,dimethoate,phenthoate,phosmet andmethidathion from fortified Chinese cabbage samples ranged between 77.1% and 104.7%,theCVs were lower than 13.81%.This assay can be used in monitoring studies for themulti-residue determination of O,O-dimethyl organophosphorus pesticides.
本文结合苏果超市的实证分析，在明确我国超市鲜活食品质量安全存在问题的基础上，探讨我国超市鲜活食品质量安全体系的建设以及HACCP管理体系在超市叶菜类蔬菜供应链中的应用。此外，为提高农药残留检测技术水平，本文以甲氧基有机磷农药的共有结构O，O-二甲基硫代磷酸酯为目标检测基团，自主设计并合成了三种具有不同连接臂结构的半抗原，与牛血清白蛋白偶联作为免疫原免疫新西兰大白兔，制备广谱特异性抗体。通过对不同抗体—包被原组合筛选以及试验条件的优化，建立了甲氧基有机磷农药的广谱特异性免疫检测方法，并研究了抗原异源性对抗体亲和性以及方法灵敏度的影响。获得以下主要研究结果：
(1)在调研和分析我国超市鲜活食品质量安全现状及存在问题的基础上，对我国超市鲜活食品供应链质量安全体系的建设进行了探讨，包括设置独立的鲜活食品质量安全管理机构，加大生产基地的培育、冷链设施的投入以及管理力度；制定详细的企业操作标准规范，规范供应链每一步实施的行为；建立超市内控与第三方检验相结合的检测体系：完善超市鲜活食品溯源体系；建立由政府部门、行业主管部门、媒体以及消费者对超市鲜活食品质量安全进行监督的体系，最终形成具有推广价值的超市鲜活食品供应链质量安全体系。
(2)对HACCP管理体系在超市叶菜类蔬菜供应链的应用进行了研究。确定超市叶菜类蔬菜供应链中关键控制点，对关键控制点制定关键限值，设置相关的监控程序以及纠偏措施，并提出相应的政策措施建议。
(3)以甲氧基有机磷农药的共有结构O，O-二甲基硫代磷酸酯为目标检测基团，自主设计并合成了三种具有不同连接臂结构的半抗原，经~1HNMR、~(13)CNMR以及MS鉴定，合成成功。将三种半抗原分别通过两种方法偶联于载体蛋白上，制备免疫原与包被原，经紫外光谱以及红外光谱鉴定，偶联成功。
(4)抗原异源性对抗体亲和性以及免疫方法灵敏度影响的研究结果显示：抗原异源会降低由简单连接臂抗原产生的抗体对包被原的吸附作用，降低抗体的亲和性，但是会增加由复杂连接臂抗原产生的抗体对包被原的吸附作用，增加抗体的亲和性，这可能由于偶联方法不同所引起偶联比高低不同，以及不同的反应试剂对蛋白质空间构象影响不同所造成；抗原异源性对于提高由复杂连接臂抗原产生的抗体的灵敏度具有比较重要的作用，但对于由简单连接臂抗原产生的抗体的灵敏度作用则相反。具有简单连接臂的抗原较复杂连接臂抗原更适合产生灵敏度高的抗体。
(5)通过对免疫原免疫新西兰大白兔获得的抗体进行筛选以及试验条件的优化，建立了甲氧基有机磷农药广谱特异性免疫检测方法。方法最佳试验条件：抗体3055稀释倍数为1：6400，包被原Ⅱ-OVA~a浓度为2μg／mL，反应介质为5％甲醇的PBS溶液，pH为6．0～7．4，溶液离子强度为0．01mol／L。方法获得的抗体亲和力以及灵敏度高，对马拉硫磷、乐果、稻丰散、亚胺硫磷、杀扑磷、杀螟硫磷、甲基对硫磷、倍硫磷等多种甲氧基有机磷农药均有较强的识别作用，其IC_(50)分别为30．1、28．9、88．3、159．7、191．7、324．0、483．9和788．9 ng／mL。应用该方法对白菜中马拉硫磷、乐果、稻丰散、亚胺硫磷以及杀扑磷的回收率进行检测，回收率位于77．1～104．7％之间，变异系数≤13．81％。
Nowadays,the food safety problems related to the fresh food have always broken out inChina.The issue of the fresh food safety,especially for organophosphorus pesticide residues,poses threat to the consumers' health.As the end of the food supply chain,the supermarketshave becoming the main channel for fresh food distribution.The food quality and safetymanagement of supermarket is at the primary stage because of the late starting.Therefore,theresearch on the quality and safety system for fresh food of supermarket and the method fordetermination of pesticides do not only have their theoretical significance,but also providessome practical advices on the quality and safety management for fresh food,and offers thequick,simple and economical method for the determination of pesticides.
The paper clarified the problems of the fresh food management of supermarket throughthe analysis of Suguo supermarket.Basing on the economic analysis of the quality and safetyfor fresh food and the investigation of the fresh food quality and safety management ofdomestic supermarkets,the construction of fresh food quality and safety system ofsupermarket in China was discussed and the application of HACCP in the leaf vegetablesupply chain of supermarket was also researched in the paper.Beside these,in order toimprove the method for the determination of pesticides,the O,O-dimethylphosphorodithioate,which is the generic structure of methoxy organophosphorus pesticide,was used as the target determinate group to generate broad specificity polycolonal antibodies.Three haptens with different spacer-arm were synthesized and conjugated to BSA to be usedas immunogens to produce the antibodies.The broad specificity immunoassay was developedthrough selecting the combinations of antibody-coating antigen and optimizing the conditionsof experiment.The influence of the antigen heterology for the affinity of antibody and thesensitivity of the method was also studied.
Through the research of this article,some beneficial conclusions have been drawn:
(1)Basing on the analysis of Suguo supermarket and the investigation of fresh foodquality and safety management of supermarket,the construction of food quality and safetysystem of supermarket in China was discussed.The food safety management organizationshould be set independently.Begin with the cultivating of the production base,the manage measures should be established separately for the three mode of fresh food supply chains,including“Production base-Supermarket”,“Production base-Merchant-Supermarket”and“Production base,Farmers' corporate organization,individual farmer-Wholesalemarket-Supermarket”.The devotion of equipments for cool chain should be increased and thepower of management should be improved.The standard of operation in every tache of thesupply chain should be done to standardize the action.The advanced method should beintroduced to develop the determination platform.The determination system should be set upby combining the supervision from supermarket and the determination from the third party.The system of tracing to the source basing on the development of technic and the databaseshould be developed.The supervision system should be constructed in every tache of thesupply chain supervised by the govemment,trade department,media and consumers.Finally,the fresh food quality and safety system of supermarket which is valuable for popularizationis formed.
(2)The application of HACCP in the leaf vegetable supply chain of supermarket wasstudied according to the implement process of HACCP.Basing on the results of hazardanalysis,the critical control points were the tache of determination before carrying away fromproduction base and the tache of acceptance by supermarket.The critical limites of pesticides,heavy metals and sense inspection,the inspection procedure and the correction measure werealso established.Some correlative policy measures were recommended.
(3)The O,O-dimethyl phosphorodithioate which is the generic structure of methoxyorganophosphorus pesticide was used as the target determinate group.Three haptens withdifferent spacer-arms were synthesized.~1HNMR,~(13)CNMR and MS spectra data supported allstructures of target molecules.The haptens were conjugated to carrier proteins forimmunogens and coating antigens by two coupling methods.UV-vis and IR spectral dataconfirmed the structures of the final conjugates.
(4)The effect of antigen heterology on antibody affinity and immunoassay sensitivitywas also studied.The results showed as follows:The titer difference between homologousand heterologous was great.The heterology in spacer arm may be not available for theantigen recognition by antibodies generated from immunogen with simple spacer arm.Thetiter difference may be due to the different coupling methods resulting in different haptenloads and different influence in the space structure of protein.Antigen heterology is moreimportant for the antiserum immunized by the hapten with complicated spacer arm than thesimple spacer arm to improve the sensitivity of immunoassay.The hapten with simple spacerarm may be more conformable to produce the antibody with high sensitivity.
(5)A general and broad class-specific enzyme-linked immunosorbent assay was developed for the O,O-dimethyl organophosphorus pesticides.The optimized conditions ofassay used antibody 3055 at a dilution of 1:6400,coating antigenⅡ-OVA~a at 2μg/mL,andanalyte in 5% methanol-0.01 mol/L PBS buffer,pH is between 6.0～7.4.The IC50 values ofthe assay,under optimized conditions,were estimated to be 30.1 ng/mL for malathion,28.9ng/mL for dimethoate,88.3 ng/mL for phenthoate,159.7 ng/mL for phosmet,191.7 ng/mL formethidathion,324.0 ng/mL for fenitrothion,483.9 ng/mL for methyl parathion,and 788.9ng/mL for fenthion.Recoveries of malathion,dimethoate,phenthoate,phosmet andmethidathion from fortified Chinese cabbage samples ranged between 77.1% and 104.7%,theCVs were lower than 13.81%.This assay can be used in monitoring studies for themulti-residue determination of O,O-dimethyl organophosphorus pesticides.
引文
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