李向梅1,2 ，王战辉1，肖希龙1，王照鹏2，温凯1，吴小平2，夏曦1，武晋孝3，江海洋1

（1中国农业大学动物医学院，北京 100193；2北京维德维康生物技术有限公司，北京 100095；3山西省饲料兽药监察所，山西 030027）

摘要：【目的】喹诺酮类药物和庆大霉素均为、广谱抗生素，对大多数革兰氏阳性菌和革兰氏阴性菌都具有显著的抗菌效果，是中国畜牧业和水产业中常用的两类兽药。由于这两类药物在动物源性食品中的残留可能导致对人类健康的危害，因此，为了保护消费者的健康，研究和制定动物源性食品中同时检测这两类药物的残留检测方法对完善中国的食品安全监测体系具有重要意义。【方法】建立了同时检测牛奶中13种喹诺酮类和庆大霉素残留的胶体金免疫层析方法。利用获得的喹诺酮类和庆大霉素单克隆抗体，采用柠檬酸三钠还原法制备胶体金颗粒，并对这两类抗体按比例进行混合标记。同时，采用方阵法系统研究了胶体金标记这两类抗体时的pH值和抗体用量对灵敏度的影响，并对这两类药物抗原的包被条件进行选择确定。在此基础上研发出可同时检测牛奶中13种喹诺酮类药物和庆大霉素的胶体金快速检测试纸条，试纸条采用直接竞争法原理。【结果】该方法可同时检测恩诺沙星、环丙沙星、诺氟沙星、氟甲喹、培氟沙星、氧氟沙星、依诺沙星、噁喹酸、麻保沙星、氟罗沙星、奥比沙星、达氟沙星和洛美沙星这13种喹诺酮类药物和庆大霉素，对其他喹诺酮类药物如：沙拉沙星、二氟沙星、司帕沙星、帕珠沙星等无交叉反应，同时对其他氨基糖苷类药物如：链霉素、新霉素、卡那霉素也无交叉反应。该试纸条对牛奶中这13种喹诺酮类药物和庆大霉素的检测限均为20 ng·mL-1，*国家对这两类药物的残留*。牛奶样本直接检测，无需处理，整个检测过程5 min内完成。【结论】采用该方法和HPLC-MS/MS对60份牛奶盲样进行对比检测，阳性样品全部检出，同时筛选方法未出现假阳性和假阴性现象，二者的测定结果基本相符，表明该方法准确可靠，适用于现场大批量样本的快速检测和筛选。实际操作过程中，可以采用胶体金免疫层析对样品进行现场快速初筛；筛选的疑似阳性样品，可以采用HPLC-MS/MS方法对样品中QNS和GEN的含量进一步确认。

关键词：喹诺酮类；庆大霉素；残留；胶体金免疫层析

Development of a Colloidal Gold Immunochromatographic Technique for Simultaneous Detection of Quinolones and Gentamicin in Milk

LI Xiang-mei1,2，WANG Zhan-hui1，XIAO Xi-long1，WANG Zhao-peng2，WEN Kai1，WU Xiao-ping2， XIA Xi1，WU Jin-xiao3，JIANG Hai-yang1

(1College of Veterinary Medicine, China Agricultural University, Beijing 100193；2Beijing WDWK Biotechnology Company, Ltd., Beijing 100095；3Shanxi Institute of Feed and Veterinary Drugs Control, Shanxi 030027)

Abstract:【Objective】Quinolones and gentamicin are highly effective and broad-spectrum antibiotics. They have significant antibiotic effects on gram-negative and gram-positive bacteria, and are widely used in agriculture in China. Because these two types of drug residues in foods of animal origin may cause harm to human health, therefore, in order to protect the consumers’ health, it is necessary to develop a detection method for simultaneous monitoring these two types of drugs residue level in food. A colloidal gold immunochromatographic method was developed for the simultaneous detection of 13 quinolones and gentamicin residues in milk.【Method】In this study, based on the quinolones and gentamicin monoclonal antibodies, the colloidal gold particles were prepared by sodium citrate reduction method, and mixed labeled with same ratio of these two types of monoclonal antibodies. The effect of pH and antibody amount for gold-antibody conjugation on the strip test sensitivity was investigated. Meanwhile, the coating condition of these two types of antigens was selected. A colloidal gold rapid test strip was developed to simultaneously detect 13 quinolones and gentamicin residue in milk on these bases, and the test strip using the principle of direct competition.【Result】The results showed that the method can simultaneously detect 13 quinolones and gentamicin. These 13 quinolones include enrofloxacin, ciprofloxacin, norfloxacin, flumequine, pefloxacin, ofloxacin, enoxacin, oxolinic acid, marbofloxacin, fleroxacin, orbifloxacin, danofloxacin and lomefloxacin. The test strip has no cross-reaction to other quinolones such as sarafloxacin, difloxacin, sparfloxacin and pazufloxacin, etc. At the same time, it has no cross-reaction to other aminoglycosides such as streptomycin, neomycin and kanamycin. The limit of detection was estimated to be 20 ng·mL-1 in milk for both the 13 quinolones and gentamicin, since the detection test line on the strip test compley disappeared at this concentration. The detection limit for milk sample of these two types of drugs fully meets the detection limit requirements of China. Samples were detected directly without treatment, and the entire testing process was completed within 5 min.【Conclusion】A parallel analysis of quinolones and gentamicin in 60 blind raw milk samples conducted by HPLC-MS/MS showed comparable results to those obtained from the strip test. All positive samples were detected while false positive and false negative phenomenon did not appear with this screening method. The results demonstrated that the developed method is suitable for the onsite determination of quinolones and gentamicin residues in a large number of samples. Since this method provides only qualitative and semiquantitative results, the determined positive samples should be further confirmed by more sensitive methods such as HPLC-MS/MS.

Key words: quinolones; gentamicin; residue; colloidal gold immunochromatographic

0 引言

【研究意义】喹诺酮类（QNS）和庆大霉素（GEN）均为广谱、抗生素，对大多数革兰氏阳性菌和革兰氏阴性菌都具有显著的抗菌效果，因此是农业、畜牧业和水产业中常用的兽药，也常被添加到饲料中促进动物的生长发育[1-2]。随着这两类药物在畜牧养殖业的不断应用，其残留问题日趋严重，在动物性食品中的残留不仅危害人的身体健康，如QNS可引起恶心，呕吐，影响软骨发育，GEN具有耳毒性和肾毒性，还污染环境、影响新药开发和临床用药，而且不利于养殖业的健康发展及畜禽产品的出口；更为严重的是动物性食品中残留较低浓度的药物容易诱导人类致病菌产生耐药性，不利于该类药物对人类疾病的治疗。为严格控制这两类抗生素在动物源食品中的残留，保证人民身体健康，保证进出口产品质量，提高中国畜产品的信誉，中国*第235号公告对这两类药物在动物源性食品中的zui高残留*（MRLs）均作了明确规定[3]。因此，研究这两类药物的残留检测方法对中国的食品安全监测具有非常重要的意义。【前人研究进展】目前国内外用于动物源食品中QNS和GEN残留检测的分析方法主要有微生物法[4]、放射化学法[5]、免疫分析法[6-10]（包括放射免疫法、酶联免疫法、荧光免疫法、免疫传感器和蛋白芯片）、薄层色谱法[11]、毛细管电泳色谱法[12-13]、液相色谱法[14-16]、气相色谱-质谱法[17]、液相色谱-质谱法[18-20]等。微生物方法所需设备简单、价格低廉, 适合于大批样品的检测；但是该法存在菌种不易筛选, 影响因素复杂、检测耗时长、稳定性差、度低、灵敏度低的特点, 往往不能满足目前实际检测工作的要求。液相色谱、质谱联机等仪器方法灵敏度高，可以提供定量及确证分析，但是前处理复杂，需要昂贵的仪器，且对操作人员有专业的要求，不适合基层检测分析。【本研究切入点】免疫分析法是近年来快速发展的一种方法, 具有灵敏度高、特异性强、适用范围广、操作简便、快捷、成本低廉及现场适应能力强等优点。胶体金免疫层析法与其他免疫方法如ELISA 相比，样品前处理简单，不需任何仪器，结果可在5—10min内获取，且对操作人员没有专业要求，非常适合现场大批量样本的筛选。但目前国内外还未出现可同时检测QNS和GEN两类药物的胶体金免疫方法的报道。【拟解决的关键问题】建立同时检测牛奶中QNS和GEN两类药物残留的胶体金免疫层析方法。