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饲料中硝基呋喃类药物检测的胶体金试纸条研制

Study on Rapid Testing Strip for Nitrofurans in Feeds

作者: 专业:兽医(专业学位) 导师:吴国娟 年度:2015 学位:硕士  院校: 北京农学院

Keywords

nitrofurans drug, residue detection, colloidal gold, strip, feed

        硝基呋喃类药物是一类人工合成的具有5-硝基呋喃环母核的广谱抗菌药物,主要有呋喃西林、呋喃妥因、硝呋烯腙、呋喃唑酮、呋喃它酮、呋喃苯烯酸钠、硝呋索尔等。这类药物抗菌谱广泛,效高价廉,被广泛用于畜禽、水产动物疾病的预防和治疗。据研究表明,该类药物对畜禽具有毒性,也是一类具有潜在致癌和致突变物质。硝基呋喃类药物在动物性食品中的残留问题对人类健康造成严重威胁,导致世界各国禁止使用。目前,国内外对硝基呋喃类药物的检测方法主要是HPLC、LC-M、LC-MS/MS、ELISA等,且这些检测方法主要是检测动物性食品中的硝基呋喃类代谢物,对于饲料中检测硝基呋喃类药物原药的文献报道不多。这些检测方法精确度高、需要专门的仪器、时间长,成本高,需要专业人员操作,不能实施在线检测,不易普及。而胶体金试纸条是以抗原抗体特异性结合为基础,具有快速、灵敏度高,不需要专业人员操作,成本低等特点,具有广阔的应用前景。本研究旨在研发出一种检测畜禽饲料中硝基呋喃类药物的胶体金试纸条,从动物性食品源头上控制残留,消除硝基呋喃类药物残留对人体的危害。本研究以5-硝基呋喃-2-甲醛为模板合成硝基呋喃类药物的共有半抗原,分别采用CDI法和NHS酯法合成了两种免疫原和包被原。两种免疫原分别免疫Balb/c小鼠各10只,免疫后采血检测,挑选血清效价高、抑制率平均的小鼠取脾脏进行细胞融合制备单抗,成功获得针对硝基呋喃类药物的抗体。纯化后的腹水效价为1:1.8×106,对呋喃西林、呋喃唑酮、呋喃妥因、呋喃它酮、呋喃苯烯酸钠、硝呋索尔、硝呋烯腙IC50分别为0.339ppb、0.462ppb、0.304ppb、0.535ppb、0.486ppb、 0.578ppb、0.578ppb。用柠檬酸钠还原法制备出粒径为20nm、30nm、40nm的胶体金颗粒,并对这几种粒径的胶体金颗粒进行了一系列实验筛选,最终确定使用30nm粒径的胶体金。通过筛选NC膜、金标垫、样品垫、吸水纸等,确定了最佳耗材。对所研制的试纸条进行了特异性、敏感性实验,该产品仅与硝基呋喃类药物发生特异性反应,与BSA、克伦特罗等其他药物不发生特异性反应,试纸条检测呋喃西林、呋喃唑酮、呋喃妥因、呋喃它酮、呋喃苯烯酸钠、硝呋索尔、硝呋烯腙检测限分别为30ng/ml、40ng/ml、30ng/ml、50ng/ml、40ng/ml、50ng/ml、80ng/ml、以直接稀释法对饲料样本进行处理,进行添加回收实验,并通过ELISA和HPLC两种检测方法验证试纸条的有效性。批间、批内重复性良好,通过3个月老化试验结果表明,可保证检测结果的稳定性,可成功的应用于饲料中硝基呋喃类抗生素的检测。该试纸条检测方法无需仪器,操作简单、快速,10min可读结果,实验结果稳定性高,可做畜禽饲料中硝基呋喃类药物现场大量筛选的有效手段,也可为其他农兽药残留检测试纸条的研制提供借鉴。
    Nitrofurans drug is a kind of synthetic with 5-nitrofuran ring mother nucleus of antibacterial drugs, mainly includes nitrofurazone, nitrofurantoin, nitrovin, furazolidone, furaltadone, nifurstyrenate sodium, nifursol, nifuroxazide, etc.The drugs,which are broad antibacterial spectrum, high efficiency and low price, arewidely used in livestock, poultry and aquatic animal disease prevention and treatment. According to the previous study, the nitrofurans drug for livestock and poultry has toxicity, and is also a kind of potentially carcinogenic and mutagenic substances.Nitrofurans drug residues in animal food have potential health riskathuman.Therefore, Nitrofurans ban the use of world.At present, the detection method of nitrofurans drug mainly HPLC, LC-MS, LC/MS/MS and ELISA, and the detection methods are mainly nitrofurans metabolites in animal food.There are few literatures reported in detection of nitrofurans drug technical in feed. The high precisiondetection methods used nowrequire specialized equipment, and the test time is long, not easy to popularize. However, the colloidal gold strip is based on the antigen antibody specificity, hashigh sensitivityand the test time is fast,and don’t need professional operation,and so on.So the colloidal gold striphas a broad application prospect.The aim of this study is to develop a kind of detection,which can detect the nitrofurans drug in livestock and poultry feed, andcompletely eliminate nitrofurans drug harm to human body.In this study,we used 5-nitrofuran-2-formaldehyde astemplate,then synthesiednitrofurans drug of haptensusing the methods of CDI and NHS respectively.We immuned10 Balb/c mouseused those two immunogen respectively.After the immune blood testing, we select the high titerserumto preparation of single cell fusion of the spleen. After purification of ascites titer of1:1.8×106. Nitrofurazone IC50 was0.339ppb, furazolidone IC50 was0.462ppb, nitrofurantoin IC50 was0.304ppb, furaltadone IC50 was0.535ppb, nifurstyrenate sodium IC50 was0.486ppb, nifursol IC50 was0.578ppb, and nitrovin IC50 was0.578ppb.The particle sizes prepared by sodium citrate reduction method were20 nm,30 nm,40 nm colloidal gold particles, we finally chose to use 30 nm size of colloidal gold, after doing a series of colloidal gold particles selectionexperiments. Through the screening of NC membrane, gold standard pad, sample pad, blotting paper and paired antibody, we determined the best materials. We developed the specificity and sensitivity experiments, and this product had nitrofurans drug reaction specificity, did not have specificity reactionwith BSA, clenbuterol and other drugs. The detection limit of nitrofurazone, furazolidone, nitrofurantoin, furaltadone, nifurstyrenate sodium, nifursol and nitrovin was30 ng/ml,40 ng/ml,30 ng/ml,50 ng/ml,40 ng/ml,50 ng/ml and 80 ng/mlrespectively. We did the experiments of processingsamplesby direct dilution and feed add recycling experiment, and two detection methodsby ELISA and HPLC.which validated the effectiveness of the strip. Thebetween batch and batch repeatability is good, and through 3 months aging tests, results shown that the strip can be successfully used in the feed nitrofurans antibiotics detection.The strip can be analyzed without instruments, and the operation is simple, rapid,and the results can be read in 10 minutes. The experimental results have high stability, and the strip can be used to do nitrofurans drug scene of livestock and poultry feed when a large number of screening effective means, also be used for other agricultural development of veterinary drug residue detection strip for reference.
        

饲料中硝基呋喃类药物检测的胶体金试纸条研制

摘要3-4
Abstract4
缩略词6-7
1 绪论7-19
    1.1 我国畜禽饲料中兽药残留现状7
    1.2 硝基呋喃类药物概述7-11
    1.3 硝基呋喃类药物检测现状11-14
    1.4 单克隆抗体技术概述14-15
    1.5 胶体金试纸条技术概述15-17
    1.6 本课题研究的主要内容与目标17-19
2 硝基呋喃类药物抗体的制备19-43
    2.1 实验材料与仪器19-21
    2.2 试验方法21-29
    2.3 结果与分析29-39
    2.4 讨论39-43
3 硝基呋喃类药物胶体金试纸条的制备43-70
    3.1 实验材料与仪器43-44
    3.2 试验方法44-50
    3.3 结果与分析50-66
    3.4 讨论66-70
4 结论70-71
参考文献71-77
致谢77-78
作者简历78-79
        下载全文需50


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