盐酸克伦特罗的免疫检测技术

盐酸克伦特罗的免疫检测技术 肉品中盐酸克伦特罗的危害及其免疫检测技术 题 目: 姓 名: 郑峰 学 院: 食品科学与药学学院 专 业: 食品科学 班 级: 2013级研究生 学 号: 1340020279 成 绩, 指导教师: 王子荣 职称: 教授 200 年 4月 24日 新疆农业大学教务处制 while the lawn is still in a great stage of life activities, compared with the previous two periods, lawn water requirements increase at this stage, if not timely irrigation, not only affect the growth of grass, also causes yellow. At this stage, according to 4-5 of irrigation. In short, grass types, water requirements are different, you mustAccording to the climate conditions and plant species to determine irrigation of the lawn. About a day, when irrigation is better depends on irrigation. In theory, the same period as long as the amount of irrigation water is less than the infiltration capacity of the soil, irrigation all the time in a day. Then depends on the irrigation way. If intermittent intermittent spray or spray irrigation (high degree of atomization), irrigation is best with the Sun. Not only can add moisture, but also significantly improve the microclimate and photosynthesis and transpiration and gas exchange, facilitated the coordination of water, air, fertilizer and soil thermal and vegetative organs of root and root expansion, and could lead the entire transit storage of the plant material, breathing, lawns and lush. If using water, irrigation, and so on, should be watching season, early spring, late autumn around the heel as well and the rest take twilight as much. (2) irrigation irrigation quantity should be based on the soil, growing, grass and other factors to determine. General lawn growing season drought period, takes about a week to pay 20-40mm; vigorously growing lawn in the heat and severe drought situations, need to pay 50-60mm per week or more. In General, regardless of the method, should be irrigated more than once, each time less water, maximum surface runoff that just occurred. 2, fertilizing to maintain lawn dark green leaves, grows, portions must be fertilized. Lawn plants for leaf growth and blossom-free requirements, so nitrogen is more important, response to nitrogen fertilizer is the most obvious. Should be basic application 肉品中盐酸克伦特罗的危害及其免疫检测技术 郑峰 指导老师:王子荣教授 摘要:本文介绍了盐酸克伦特罗的理化特性和生理作用，分析了它在肉制品中残 留对人体造成的危害，同时综述了肉品中残留盐酸克伦特罗的胶体金免疫检测技 术和酶联免疫检测技术的原理和操作要点，分析了两种免疫检测技术的特点，总 结了两种免疫检测技术的技术优势并提出自己的观点。 关键词:盐酸克伦特罗;生理;胶体金检测;酶联免疫 The detection of Clenbuterol hydrochloride residues Absttact:This paper describes the effects in physiology of the clenbuterol hydrochloride remained in meat products,and analyzes the harms which caused by the residues in meat; The artical also summarizes the characteristics of technology principles with gold and enzyme-linked immunoassay to analyze the residues in meat and the operation points in immunoassay technique,contrasting the two immunoassay techniques.Finally the advantages of both immunoassay techniques were summarized and views were put forward on. Key words: Clenbuterol;DIGFA;ELISA 目前食品安全问题已经成为国内最为关注的问题，其关系到每个人的将抗发 展。畜产品在人类饮食结构中占有重要的地位，如何确保畜产品的安全是目前急 需解决的问题。市面上畜肉产品的安全卫生主要由两方面因素决定，即宰前因素 和宰后因素，宰前因素主要是由动物的养殖方式和病害控制对肉品质产生的影 响;宰后因素主要现在宰后检验、运输、加工、包装等方面。随着一系列由饲 料问题引起的肉品安全问题的发生(二噁英、疯牛病、苏丹红和瘦肉精)，人们 渐渐认识到饲料安全在保障畜产品安全中的重要性。饲料安全性关系到畜产品的 质量与人类的健康。 盐酸克伦特罗(Clenbuterol Hydrochloride，CL)俗称瘦肉精是国家违禁药 [1]物中的一种。盐酸克伦特罗是一种β-肾上腺素受体激动剂，曾作为药物用于治 while the lawn is still in a great stage of life activities, compared with the previous two periods, lawn water requirements increase at this stage, if not timely irrigation, not only affect the growth of grass, also causes yellow. At this stage, according to 4-5 of irrigation. In short, grass types, water requirements are different, you mustAccording to the climate conditions and plant species to determine irrigation of the lawn. About a day, when irrigation is better depends on irrigation. In theory, the same period as long as the amount of irrigation water is less than the infiltration capacity of the soil, irrigation all the time in a day. Then depends on the irrigation way. If intermittent intermittent spray or spray irrigation (high degree of atomization), irrigation is best with the Sun. Not only can add moisture, but also significantly improve the microclimate and photosynthesis and transpiration and gas exchange, facilitated the coordination of water, air, fertilizer and soil thermal and vegetative organs of root and root expansion, and could lead the entire transit storage of the plant material, breathing, lawns and lush. If using water, irrigation, and so on, should be watching season, early spring, late autumn around the heel as well and the rest take twilight as much. (2) irrigation irrigation quantity should be based on the soil, growing, grass and other factors to determine. General lawn growing season drought period, takes about a week to pay 20-40mm; vigorously growing lawn in the heat and severe drought situations, need to pay 50-60mm per week or more. In General, regardless of the method, should be irrigated more than once, each time less water, maximum surface runoff that just occurred. 2, fertilizing to maintain lawn dark green leaves, grows, portions must be fertilized. Lawn plants for leaf growth and blossom-free requirements, so nitrogen is more important, response to nitrogen fertilizer is the most obvious. Should be basic application 疗人的呼吸系统疾病(哮喘等)，1984年被发现其具有提高动物胴体瘦肉率的效果，所以CL被违规添加到动物饲料中，尤其在猪的养殖中。饲料中添加CL喂养畜禽会导致其在动物组织中残留蓄积，食用此类的肉制品会引起急性或慢性的 [2]中毒反应。因此，世界上许多国家都禁止在畜禽的饲养中使用CL，中国农业部1997年发文禁止瘦肉精在饲料和畜牧生产中使用，商务部自2009年12月9日起，禁止进出口盐酸克伦特罗，2001年12月27日—4月9日，农业部分别下 [1]发文件禁止使用β-激动剂类药物作为饲料添加剂。但是受到利益的驱使，CL的滥用现象仍屡禁不止。2011年3月15日河南“瘦肉精”案件被央视曝光，不仅使公众持续高度关注，也暴露出监管中出现的问题。 1 盐酸克伦特罗的理化特性及作用机理 2.1 盐酸克伦特罗的理化特性 盐酸克伦特罗(Clenbuterol，CL)，俗名瘦肉精，是β-肾上腺素受体激动剂的2 [3]一种，呈白色结晶状，略苦，易溶于水、甲醇、乙醇等溶剂，不易溶于氯仿，其化学性质比较稳定，超高温度下才能发生缓慢分解现象。研究表明，含有CL的畜禽产品经100?沸水煮过后，所含该药的残留物基本不变。动物食用后在消 起效，2.5h后血液浓度达到峰值，其作用时间化吸收系统内迅速吸收，20min内 长达5h，消除半衰期时间较长，一个半衰期大约一天，其主要残留在动物眼睛、 [4]毛发和内脏中，其中动物肝脏中残留最多。在CL的应用数年内国际上发生多起食用残留CL肉制品的中毒事件，人们逐渐注意到CL的毒副作用。 1.2 CL的作用机理 CL作为一种β-受体激动剂，起到拟交感神经的作用。最初，它作为治疗疾2 病的药剂被使用，医学研究表明其可用于治疗支气管炎、哮喘性慢性支气管炎以 [4]及呼吸系统疾病所导致的支气管痉挛。这是由于其具有松弛支气管肌肉的作用，明显增加支气管纤毛运动促进粘液溶解，有利于病人痰液的顺利排出。 其生化机理表现在CL首先能促使细胞内环状腺苷酸(cAMP)的合成，激活cAMP参与的蛋白激酶，被激活的激酶在细胞脂肪组织内可以抑制乙酰辅酶A、羟化酶和甘油三酯合成酶的活性，提高了脂肪分解酶的活性，参与调节三羧酸循 [5]环，加快脂肪的分解。CL在动物饲养中提高了动物胴体品质，使瘦肉率增加 另一方面，CL可以降低动物血液中的12%左右，骨骼肌内脂肪降低10%左右。 +Mg的浓度提高肌肉的兴奋度，并改变细胞膜的通透性，抑制脂肪的合成和蛋白while the lawn is still in a great stage of life activities, compared with the previous two periods, lawn water requirements increase at this stage, if not timely irrigation, not only affect the growth of grass, also causes yellow. At this stage, according to 4-5 of irrigation. In short, grass types, water requirements are different, you mustAccording to the climate conditions and plant species to determine irrigation of the lawn. About a day, when irrigation is better depends on irrigation. In theory, the same period as long as the amount of irrigation water is less than the infiltration capacity of the soil, irrigation all the time in a day. Then depends on the irrigation way. If intermittent intermittent spray or spray irrigation (high degree of atomization), irrigation is best with the Sun. Not only can add moisture, but also significantly improve the microclimate and photosynthesis and transpiration and gas exchange, facilitated the coordination of water, air, fertilizer and soil thermal and vegetative organs of root and root expansion, and could lead the entire transit storage of the plant material, breathing, lawns and lush. If using water, irrigation, and so on, should be watching season, early spring, late autumn around the heel as well and the rest take twilight as much. (2) irrigation irrigation quantity should be based on the soil, growing, grass and other factors to determine. General lawn growing season drought period, takes about a week to pay 20-40mm; vigorously growing lawn in the heat and severe drought situations, need to pay 50-60mm per week or more. In General, regardless of the method, should be irrigated more than once, each time less water, maximum surface runoff that just occurred. 2, fertilizing to maintain lawn dark green leaves, grows, portions must be fertilized. Lawn plants for leaf growth and blossom-free requirements, so nitrogen is more important, response to nitrogen fertilizer is the most obvious. Should be basic application 质的分解，从而使大量的氮转化为蛋白质(瘦肉)。 其作用图如下: 克伦特罗 ? 受体?修饰受体 ? 腺苷酸环化酶(无活性)?腺甘酸环化酶(无活性)蛋白质激酶(无活性) ? ? ATP ? cAMP ? 蛋白质激酶(有活性) 激素敏感酯酶(无活性)?? 激素敏感酯酶(有活性) 甘油三酯?? [5]脂肪酸+甘油 2 盐酸克伦特罗的危害 少量的盐酸克伦特罗可以用于治疗呼吸道疾病，大量使用CL对动物有害，如果动物饲料中含有CL，动物会表现为:血管急剧扩张、心率加快、呼吸速度加快、体温的上升、心脏和肾脏负担加重等反应，长期将其喂食动物会大量减少机体内受体数量，对CL敏感性降低，导致动物自身体内促肌肉生长激素减少从 [6]而自生生长效应逐渐丧失。 人食用了含有CL残留的肉制品会出现类似于动物甚至比动物更严重的症状，人类使用超过的肉后会出现急性中毒症状发生头痛、头晕、乏力、胸闷、心悸、骨骼肌震颤、四肢麻木等一系列不良反应。血液检查可见白细胞增多、血钾偏低、血液中丙酮酸含量升高。长期食用含有盐酸克伦特罗的肉和内脏会引起人体心血管系统和神经系统的疾病，降低血管壁弹性、血管周围的神经被扩张的血管压迫产生心肌收缩加强、心跳加快、呼吸加剧等不良反应，严重了还能引起机体代谢紊乱。 研究表明，盐酸克伦特罗能使大鼠SOD活性显著降低，MDA含量明显升高，使大鼠胸腺和脾脏发生一系列的病理损伤，说明该物质能破坏动物的免疫器官， [7]对动物机体有较大的危害。 3免疫分析技术在检测肉品中残留盐酸克伦特罗的应用 while the lawn is still in a great stage of life activities, compared with the previous two periods, lawn water requirements increase at this stage, if not timely irrigation, not only affect the growth of grass, also causes yellow. At this stage, according to 4-5 of irrigation. In short, grass types, water requirements are different, you mustAccording to the climate conditions and plant species to determine irrigation of the lawn. About a day, when irrigation is better depends on irrigation. In theory, the same period as long as the amount of irrigation water is less than the infiltration capacity of the soil, irrigation all the time in a day. Then depends on the irrigation way. If intermittent intermittent spray or spray irrigation (high degree of atomization), irrigation is best with the Sun. Not only can add moisture, but also significantly improve the microclimate and photosynthesis and transpiration and gas exchange, facilitated the coordination of water, air, fertilizer and soil thermal and vegetative organs of root and root expansion, and could lead the entire transit storage of the plant material, breathing, lawns and lush. If using water, irrigation, and so on, should be watching season, early spring, late autumn around the heel as well and the rest take twilight as much. (2) irrigation irrigation quantity should be based on the soil, growing, grass and other factors to determine. General lawn growing season drought period, takes about a week to pay 20-40mm; vigorously growing lawn in the heat and severe drought situations, need to pay 50-60mm per week or more. In General, regardless of the method, should be irrigated more than once, each time less water, maximum surface runoff that just occurred. 2, fertilizing to maintain lawn dark green leaves, grows, portions must be fertilized. Lawn plants for leaf growth and blossom-free requirements, so nitrogen is more important, response to nitrogen fertilizer is the most obvious. Should be basic application 免疫检测分析技术是建立在免疫学基础上的一项检测技术，以抗原抗体特异性结合反应为基础原理。为检测特异性的微生物及其毒素，相应抗体被广泛用于 [8]免疫检测方法中。其中大部分抗体都来源于鼠或兔血清，其主要指标包括 单克隆抗体。多克隆抗体因为在制备过程中特异性和灵敏性，分为多克隆抗体与 操作较简单、制备周期较短，因此，在免疫学检测中应用的比例较大。但多克隆抗也有其自身缺点，由于动物免疫反应具有可变性和偶然性，所以特异性方面没有单克隆抗体可靠、稳定，免疫检测技术由于其操作方便，出结果较快所以被应 [9]用于肉品种CL残留量的检测。免疫分析技术主要包括滴金免疫技术(DIGFA) 、酶联免疫技术(ELISA)、生物免疫传感器技术(BS)和放射免疫分析技 [8]术(RIA)等。 3.1胶体金检测盐酸克伦特罗 3.1.1 原理: 胶体金法检测原理是样品在检测卡上从加样孔开始依次向检测线(T 线)、质控线(C 线)泳动。加入CL样品后，金标 CL 单抗体会被 T 线上固定化的 CL 捕获，形成一条棕红色的 T 线;此时过量的金标 CL 单抗继续涌动被 C 线 [10]上固定化的二抗捕获形成一条棕红色的 C 线。如果样品呈阳性，CL 将与固定化在 T 线上的人工抗原竞争有限的胶体金标记单抗，样品中CL含量越多，那么检测线的颜色越浅。 3.1.2 检测技术 将样本约 4g 绞碎装入 5mL 的离心管，90?以上水浴加热10min 后取出冷却至室温。吸取2-3滴PBST 和样品渗出液到1.5mL离心管混合后，取 3 滴 [11]滴加于加样孔中， 加3滴 8ppb 的甲醇克伦特罗，4-5min 后读取结果。 3.1.3 胶体金法检测特点 以胶体金标记盐酸克伦特罗单克隆抗体竞争性膜层析技术检测畜肉中盐酸克伦特罗的残留。如果待测样中无盐酸克伦特罗，则金标抗体能顺利地与固相载 [12]体结合，形成肉眼可见的红色带;若待测样中含有盐酸克伦特罗，则与金标抗体以同样的机会与固相载体结合，竞争性地占去了金标抗体与固相载体结合的机会，使金标抗体与固相载体的结合量减少，无法形成肉眼可见的红色带。胶体金免疫层析技术是将克伦特罗的抗体与胶体金结合形成免疫金，制成检测结果肉眼可见的试纸条。此法有特异性好、灵敏度高、简便、快速、对实验条件和操作while the lawn is still in a great stage of life activities, compared with the previous two periods, lawn water requirements increase at this stage, if not timely irrigation, not only affect the growth of grass, also causes yellow. At this stage, according to 4-5 of irrigation. In short, grass types, water requirements are different, you mustAccording to the climate conditions and plant species to determine irrigation of the lawn. About a day, when irrigation is better depends on irrigation. In theory, the same period as long as the amount of irrigation water is less than the infiltration capacity of the soil, irrigation all the time in a day. Then depends on the irrigation way. If intermittent intermittent spray or spray irrigation (high degree of atomization), irrigation is best with the Sun. Not only can add moisture, but also significantly improve the microclimate and photosynthesis and transpiration and gas exchange, facilitated the coordination of water, air, fertilizer and soil thermal and vegetative organs of root and root expansion, and could lead the entire transit storage of the plant material, breathing, lawns and lush. If using water, irrigation, and so on, should be watching season, early spring, late autumn around the heel as well and the rest take twilight as much. (2) irrigation irrigation quantity should be based on the soil, growing, grass and other factors to determine. General lawn growing season drought period, takes about a week to pay 20-40mm; vigorously growing lawn in the heat and severe drought situations, need to pay 50-60mm per week or more. In General, regardless of the method, should be irrigated more than once, each time less water, maximum surface runoff that just occurred. 2, fertilizing to maintain lawn dark green leaves, grows, portions must be fertilized. Lawn plants for leaf growth and blossom-free requirements, so nitrogen is more important, response to nitrogen fertilizer is the most obvious. Should be basic application 者要求不高等特点，适合用作大批量样本的初步筛选和现场检测，缺点是假阳性 [13]率较高。 3.2 酶联免疫分析技术(EIA) 3.2.1 原理 [11]?使抗原或抗体结合到某种固相载体表面，并保持其免疫活性。 ?使抗原或抗体与某种酶连接成酶标抗原或抗体，这种酶标抗原或抗体既保留其免疫活性，又保留酶的活性。在测定时，把受检标本(测定其中的抗体或抗原)和酶标抗原或抗体按不同的步骤与固相载体表面的抗原或抗体起反应。洗涤使载体上形成的抗原抗体复合物与未结合的其他物质分开，最后结合在固相载体 [11]上的酶量与标本中受检物质的量成一定的比例。加入酶反应相应的底物，底物被酶催化发生变色反应，产物生成的量与标本中受检物质的量有直接关系，所以可以根据颜色反应的变化程度来进行定性和定量分析。酶的催化率比较高，可以 [13]放大效果，从而使测定方法达到很高的敏感度。 3.2.2 检测方法 称取 2.0左右去除肥肉和筋膜的瘦肉样本绞碎，置于 10mL离心管中;加入 6mL 组织样本提取液，振荡混匀，静置约 10min;2500r/min 离心 12min;取1mL 上清液，加入20μL氢氧化钠溶液混匀后 2500r/min 离心 7min，冷藏备查。取出微孔板，对应微孔编号，每个样本做 1 孔，标准做 2 孔，阴、阳性对 [14]照各 2 孔。每孔滴加样本 20μL，再加入 50μL CL 酶标物，再加入 CL 抗体试剂 80μL 振荡混匀后置 25?避光环境中反应 30min。洗涤 5 次后加入底物液 A、B 液 50μL 振荡混匀， 25?避光环境中反应15,20min 后加入终止液 50μL 振荡混匀，于双波长 450/630nm 检测，5min 内读完每孔 OD 值 3.2.3酶联免疫分析技术特点 ELISA可用于测定抗原，也可用于测定抗体。在这种测定方法中有3种必要的试剂: [15] ?固相的抗原或抗体。 ?酶标记的抗原或抗体。 ?酶作用的底物。根据试剂的来源和标本的性状以及检测的具备条件，可设计出各种不同类型的检测方法。 while the lawn is still in a great stage of life activities, compared with the previous two periods, lawn water requirements increase at this stage, if not timely irrigation, not only affect the growth of grass, also causes yellow. At this stage, according to 4-5 of irrigation. In short, grass types, water requirements are different, you mustAccording to the climate conditions and plant species to determine irrigation of the lawn. About a day, when irrigation is better depends on irrigation. In theory, the same period as long as the amount of irrigation water is less than the infiltration capacity of the soil, irrigation all the time in a day. Then depends on the irrigation way. If intermittent intermittent spray or spray irrigation (high degree of atomization), irrigation is best with the Sun. Not only can add moisture, but also significantly improve the microclimate and photosynthesis and transpiration and gas exchange, facilitated the coordination of water, air, fertilizer and soil thermal and vegetative organs of root and root expansion, and could lead the entire transit storage of the plant material, breathing, lawns and lush. If using water, irrigation, and so on, should be watching season, early spring, late autumn around the heel as well and the rest take twilight as much. (2) irrigation irrigation quantity should be based on the soil, growing, grass and other factors to determine. General lawn growing season drought period, takes about a week to pay 20-40mm; vigorously growing lawn in the heat and severe drought situations, need to pay 50-60mm per week or more. In General, regardless of the method, should be irrigated more than once, each time less water, maximum surface runoff that just occurred. 2, fertilizing to maintain lawn dark green leaves, grows, portions must be fertilized. Lawn plants for leaf growth and blossom-free requirements, so nitrogen is more important, response to nitrogen fertilizer is the most obvious. Should be basic application [8]ELISA 是一种常用固相酶免疫测定方法，以酶或辅酶作为标记物来标记抗原或抗体，再通过酶促反应的放大作用来显示免疫学反应。ELISA 法具有选择性强，所用试剂易保存、性质稳定，结果判断客观、样品处理量大等优点。 4 胶体金免疫层析技术是将克伦特罗的抗体与胶体金结合形成免疫金，制成检测结果肉眼可见的试纸条。此法有特异性好、灵敏度高、简便、快速、对实验条件和操作者要求不高等特点，适合用作大批量样本的初步筛选和现场检测，缺点是假阳性率较高。 酶免疫分析技术是将抗原和抗体亲和反应的特异性、专一性与酶对底物的高效催化作用结合起来的固相免疫测定技术。陈继明等研究用酶免法检测饮料、尿样与脏器中克伦特罗含量，其检测结果与高效液相色谱法的检测结果大体一致。与胶体金法相比，ELISA法有特异性强、灵敏度高，可作定性与定量分析、结果更准确等特点。 根据目前国内外检测CL各种方法的进展情况以及CL滥用及残留的严峻性，建立一种克服上述各种检测方法缺点的简便、快速、精确、准确、可实现现场检测的测定CL技术势在必行。 while the lawn is still in a great stage of life activities, compared with the previous two periods, lawn water requirements increase at this stage, if not timely irrigation, not only affect the growth of grass, also causes yellow. At this stage, according to 4-5 of irrigation. In short, grass types, water requirements are different, you mustAccording to the climate conditions and plant species to determine irrigation of the lawn. About a day, when irrigation is better depends on irrigation. In theory, the same period as long as the amount of irrigation water is less than the infiltration capacity of the soil, irrigation all the time in a day. Then depends on the irrigation way. If intermittent intermittent spray or spray irrigation (high degree of atomization), irrigation is best with the Sun. Not only can add moisture, but also significantly improve the microclimate and photosynthesis and transpiration and gas exchange, facilitated the coordination of water, air, fertilizer and soil thermal and vegetative organs of root and root expansion, and could lead the entire transit storage of the plant material, breathing, lawns and lush. If using water, irrigation, and so on, should be watching season, early spring, late autumn around the heel as well and the rest take twilight as much. (2) irrigation irrigation quantity should be based on the soil, growing, grass and other factors to determine. General lawn growing season drought period, takes about a week to pay 20-40mm; vigorously growing lawn in the heat and severe drought situations, need to pay 50-60mm per week or more. In General, regardless of the method, should be irrigated more than once, each time less water, maximum surface runoff that just occurred. 2, fertilizing to maintain lawn dark green leaves, grows, portions must be fertilized. Lawn plants for leaf growth and blossom-free requirements, so nitrogen is more important, response to nitrogen fertilizer is the most obvious. Should be basic application 参考文献: [1] 王楠,周宇,董再蒸. 纳米金标记抗体分光光度法测定尿液中盐酸克伦特罗[J].理化检验 (化学分册),2013,10:1215-1218. [2] 曹巧玲,王中民,杨凯,姜德全. ELISA法和胶体金检测盐酸克伦特罗方法比较[A]. 中国化 学会.第七届全国仪器分析及样品预处理学术研讨会集[C].中国化学会:,2013:4. [3] 罗奕铭. 盐酸克伦特罗与莱克多巴胺联检速测卡的研制[J]. 现代食品科 技,2013,01:197-200. [4] 李颖,苏晓鸥,杨静. 盐酸克伦特罗在羊主要脏器中残留量消除规律的研究[J]. 中国畜牧 兽医,2013,02:217-220. [5] 冯晓阳,戴永康,张晟剑. 胶体金层析免疫法对盐酸克伦特罗的检测试验[J]. 广东化 工,2013,05:150-151. [6] 王秋平,哈婧,刘硕,张佳丽,苏萌. 盐酸克伦特罗检测方法的研究进展[J]. 河北工业科 技,2013,02:125-129. [7] 叶雪珠,王强,赵首萍,张永志,王钢军. 快速检测尿液中盐酸克伦特罗残留技术评价[J]. 浙 江农业科学,2013,05:576-579. [8] Ting Xu,Bao M.Wang,Wei Sheng,Qing X.Li,Xiao L. Shao,Ji Li.Application of an enzyme-linked immunosorbent assay for the detection of clenbuterol residues in swine urine and feeds[J]. Journal of Environmental Science and Health, Part B,2007,422:. [9] 陈曦,曹慧,徐斐,李红梅,于劲松. 盐酸克伦特罗酶标抗原的制备及其ELISA检测[J]. 江苏 农业学报,2013,02:458-460. [10] 王昱. 盐酸克伦特罗对小鼠大脑皮层ATP、SDH、ADA和LDH活性的影响[J]. 甘肃农 业大学学报,2013,02:21-25. [11] Yao Xun,Yan Panpan,Tang Qinghui,Deng Anping,Li Jianguo. Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol.[J]. Analytica chimica acta,2013,798:. [12] 汤轶伟,王硕,励建荣,孟良玉. β-兴奋剂盐酸克伦特罗残留检测方法最新研究进展[J]. 中国食品学报,2013,05:158-164. [13] 顾可飞,周昌艳,王红梅,赵志辉. 盐酸克伦特罗辐解产物对小鼠体重和心脏形态学影响 初步分析[J]. 中国食物与营养,2013,06:13-16. [14] 肖治理,余建华,雷红涛,杨金易,孙远明. 盐酸克伦特罗快速检测胶体金试纸的研制[J]. 现代食品科技,2013,08:2004-2010. while the lawn is still in a great stage of life activities, compared with the previous two periods, lawn water requirements increase at this stage, if not timely irrigation, not only affect the growth of grass, also causes yellow. At this stage, according to 4-5 of irrigation. In short, grass types, water requirements are different, you mustAccording to the climate conditions and plant species to determine irrigation of the lawn. About a day, when irrigation is better depends on irrigation. In theory, the same period as long as the amount of irrigation water is less than the infiltration capacity of the soil, irrigation all the time in a day. Then depends on the irrigation way. If intermittent intermittent spray or spray irrigation (high degree of atomization), irrigation is best with the Sun. Not only can add moisture, but also significantly improve the microclimate and photosynthesis and transpiration and gas exchange, facilitated the coordination of water, air, fertilizer and soil thermal and vegetative organs of root and root expansion, and could lead the entire transit storage of the plant material, breathing, lawns and lush. If using water, irrigation, and so on, should be watching season, early spring, late autumn around the heel as well and the rest take twilight as much. (2) irrigation irrigation quantity should be based on the soil, growing, grass and other factors to determine. General lawn growing season drought period, takes about a week to pay 20-40mm; vigorously growing lawn in the heat and severe drought situations, need to pay 50-60mm per week or more. In General, regardless of the method, should be irrigated more than once, each time less water, maximum surface runoff that just occurred. 2, fertilizing to maintain lawn dark green leaves, grows, portions must be fertilized. Lawn plants for leaf growth and blossom-free requirements, so nitrogen is more important, response to nitrogen fertilizer is the most obvious. Should be basic application [15] 李颖. 肉羊主要器官中盐酸克伦特罗检测方法及代谢残留规律的研究[D].中国农业科 学院,2013. [16]肖红玉,刘国艳,史贤明. 盐酸克伦特罗的检测方法[J]. 中国饲料,2005,12:31-33. while the lawn is still in a great stage of life activities, compared with the previous two periods, lawn water requirements increase at this stage, if not timely irrigation, not only affect the growth of grass, also causes yellow. At this stage, according to 4-5 of irrigation. In short, grass types, water requirements are different, you mustAccording to the climate conditions and plant species to determine irrigation of the lawn. About a day, when irrigation is better depends on irrigation. In theory, the same period as long as the amount of irrigation water is less than the infiltration capacity of the soil, irrigation all the time in a day. Then depends on the irrigation way. If intermittent intermittent spray or spray irrigation (high degree of atomization), irrigation is best with the Sun. Not only can add moisture, but also significantly improve the microclimate and photosynthesis and transpiration and gas exchange, facilitated the coordination of water, air, fertilizer and soil thermal and vegetative organs of root and root expansion, and could lead the entire transit storage of the plant material, breathing, lawns and lush. If using water, irrigation, and so on, should be watching season, early spring, late autumn around the heel as well and the rest take twilight as much. (2) irrigation irrigation quantity should be based on the soil, growing, grass and other factors to determine. General lawn growing season drought period, takes about a week to pay 20-40mm; vigorously growing lawn in the heat and severe drought situations, need to pay 50-60mm per week or more. In General, regardless of the method, should be irrigated more than once, each time less water, maximum surface runoff that just occurred. 2, fertilizing to maintain lawn dark green leaves, grows, portions must be fertilized. Lawn plants for leaf growth and blossom-free requirements, so nitrogen is more important, response to nitrogen fertilizer is the most obvious. Should be basic application