【作者】 倪姮佳；

【导师】 张素霞； 王战辉；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2014， 博士

【摘要】 恩诺沙星(Enrofloxacin, ENR)和磺胺二甲嘧啶(Sulfamethazine, SM2)是目前在畜牧养殖及水产业应用较广的两种抗菌药,同时也是畜产品安全着重监管的对象。目前对这两种药物的快速残留检测研究主要以抗体为结合元件建立的免疫分析方法为主。然而,抗体的制备离不开使用实验动物,而且难以重复生产,再加上其对保存条件要求严格,导致抗体的使用受到限制。核酸适配体(Aptamer)是能够与许多目标分子(蛋白,药物,无机或有机分子)发生高亲合性和特异性结合的一类单链核苷酸(DNA或RNA)。与抗体相比,核酸适配体的最大优势在于不需要使用实验动物,可用体外方法分离制备,并且其更易于修饰,也更稳定。本研究以恩诺沙星和磺胺二甲嘧啶为靶分子,首次筛选出能特异性识别这两种抗菌药的核酸适配体,并分别建立了用于牛奶中恩诺沙星和磺胺二甲嘧啶残留检测的直接竞争化学发光分析方法。通过将恩诺沙星和氧氟沙星(Ofloxacin, OFL)与磁珠(Magnetic beads, MBs)偶联,并以OFL-MBs作为反筛工具,建立了用于恩诺沙星核酸适配体筛选的磁珠辅助-指数式扩增配体的系统进化筛选体系(Mag-SELEX)。经过8轮筛选,最终得到6条ssDNA核酸适配体,分别为No.11, No.17, No.19, No.21, No.27和No.49。其中No.17、No.19和No.49的亲和力最高,其解离常数(Dissociation constant,Kd)分别为188nM、355nM和459nM。选取这三条核酸适配体进行特异性分析,结果显示No.17和No.19的特异性优于No.49。并且,No.17对恩诺沙星的结合能力最强,其对环丙沙星也有微弱的结合能力。将数据进行统计学分析,结果显示No.17具备区分恩诺沙星与环丙沙星的能力。因此,No.17将用于检测方法的建立。将磺胺二甲嘧啶和磺胺甲基嘧啶(Sulfamerazine,SMR)与磁珠偶联,并以SMR-MBs作为反筛工具,建立了用于磺胺二甲嘧啶核酸适配体筛选的Mag-SELEX体系。经过9轮筛选,共得到4条核酸适配体,分别为SAM, SA06, SA07和SA41。这四条核酸适配体都具有较高的亲和力,其解离常数分别为203nM、274nM、79nM和122nM。选取拥有较低解离常数的SA07和SA41进行特异性测定,结果显示,这两条核酸适配体均能特异性识别磺胺二甲嘧啶。因此,选择亲和力最高的SA07用于检测方法的建立。选用生物素(Biotin)化的核酸适配体biotin-No.17作为识别工具,利用链霉亲和素-生物素系统,并合成出酶标抗原ENR-spacer-HRP,建立了用于恩诺沙星残留检测的直接竞争化学发光分析法。该方法的检测限为2.26ng/mL,并且方法特异性良好,对环丙沙星的交叉反应率为2.5%。应用于牛奶样品中恩诺沙星的残留检测,其回收率为90.6-104.7%。变异系数为9.8-18.9%,结果表明本研究建立的方法可用于牛奶中恩诺沙星的残留检测。同样以生物素化的核酸适配体(biotin-SA07)为识别工具,合成出酶标抗原SM2-GA-HRP,建立了用于检测磺胺二甲嘧啶的直接竞争化学发光分析方法。该方法的检测限为0.92ng/mL,线性范围为1.85-21.57ng/mL,并且该方法对27种磺胺类药物(除了磺胺二甲嘧啶)的交叉反应率均较低。将磺胺二甲嘧啶添加至空白牛奶,其回收率为88.0-98.2%,变异系数为10.0-23.4%,以上结果表明本研究建立的方法可用于检测牛奶中磺胺二甲嘧啶的残留。更多还原

【Abstract】 Enrofloxacin (ENR) and sulfamethazine (SM2) have been wildly used to treat infections in livestock and aquatic animals. Therefore, the intensive residue monitoring for these two drugs in animal products is essential. The immunoassay, which employs antibody as the recognition element, is the most common used method for the rapid detection of veterinary drugs. However, the preparation of antibody not only needs the laboratory animals, but also requires strict storage condition. Moreover, it would be difficult to be reproduced, therefore the application of the antibody is limited. Aptamers are single-strand deoxyribonucleic acid (ssDNA) and ribonucleic acid (RNA) that can specifically bind to targets (proteins, drugs or other molecules). Compared with antibody, aptamers are selected by in vitro selection, which is an animal-friendly method. Besides, aptamers are more stable than antibodies, and they can be modified easily. In this study, ENR-specific and SM2-specific aptamers were selected by in vitro selection for the first time. And the direct competitive chemiluminescent enzyme immunoassays (dc-CLEIA) for detecting ENR and SM2were developed based on the newly developed aptamers.ENR and ofloxacin (OFL) were immobilized onto magnetic beads (MBs) by forming a covalent bond. The Mag-SELEX system was developed for selecting several aptamers that can specifically bind with ENR by introducing OFL-MBs as counter selection. After8rounds of selection, six aptamer were selected, which were No.1l, No.17, No.19, No.21, No.27and No.49. Three of them, No.17, No.19and No.49, showed the relatively high affinity towards ENR. The dissociation constants (K4) of the three aptamers were188nM,355nM and459nM, respectively. Then, the specific tests of individual aptamer were conducted among the three aptamers. The results showed that No.17and No.19were more specific to ENR than No.49did. Interestingly, No.17also exhibited a weak affinity towards cipfloxacin (CIP). But No.17was still capable to tell ENR from CEP and OFL according to the statistical analysis. Therefore, No.17will be chosen to develop the method for detection of ENR.Besides, SM2and sulfamerazine (SMR) were attached onto MBs by forming a covalent bond as well. As the tool for counter selection, SMR-MBs was introduced to develop the Mag-SELEX system for the selection of aptamers that can specifically bind to SM2. After the ninth round selection, four aptamers were selected, which were SA04, SA06, SA07and SA41. All of these aptamer showed a high affinity towards SM2, the Kd values of them were203nM,264nM,79nM and122nM separately. And the specific tests of SA07and SA41were conducted. The results showed that both aptamers were highly specific for SM2. So, SA07, the aptamer with the highest affinity, would be chosen to develop the method for the detection of SM2.The direct competitive chemiluminescent enzyme immunoassay for detecting ENR was developed by introducing the streptavidin-biotin system, the biotin-labeled No.17as the recognition element and the ENR-spacer-HRP as the enzyme-labeled antigen. The optimized method displayed a detection limit (LOD) of2.26ng/mL. And a low degree of cross-reactivity between ENR and CIP (2.5%) was observed. These results revealed that the developed method was specific for ENR. Besides, recovery of spiked milk samples with ENR was studied, the recoveries of ENR were90.6-104.7%. Assay reproducibility was satisfactory, with coefficient of variation (CV) ranging from9.8%to18.9%. These results indicated that ENR in bovine milk can be detected by this method efficiently.Meanwhile, the dc-CLEIA for the detection of SM2was also developed. The biotin-labeled SA07and SM2-GA-HRP were used in this assay as the recognition element and the enzyme-labeled antigen, respectively. The LOD values of this method was0.92ng/mL, and the working linear range was from1.85ng/mL to21.57ng/mL. The specificity test for the determination of SM2was performed by employing27SAs. And a low degree of cross-reactivity between SM2and27SAs was observed, suggesting a high specificity of the method. Besides, the analysis of SM2fortified milk samples by the developed method showed average recoveries from88.0-98.2%with CV ranged from10.0%to23.4%. Thus, these results suggested that the SA07-based dc-CLEIA was suitable for screening SM2in milk.更多还原