同时检测牛奶中喹诺酮类和庆大霉素残留的胶体金免疫层析方法研究

doi:10.3864/j.issn.0578-1752.2014.19.017

摘要/Abstract

摘要： 【目的】喹诺酮类药物和庆大霉素均为高效、广谱抗菌药物，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。采用柠檬酸三钠还原法制备胶体金颗粒，并对喹诺酮类和庆大霉素单克隆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、卡那霉素等也无交叉反应。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，完全满足国家对这两类药物的残留限量要求。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份牛奶盲样进行比对试验，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

Abstract: 【Objective】Quinolones and gentamicin are highly effective and broad-spectrum antibacterial drugs. 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, etc. 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 completely 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

李向梅，王战辉，肖希龙，王照鹏，温凯，吴小平，夏曦，武晋孝，江海洋. 同时检测牛奶中喹诺酮类和庆大霉素残留的胶体金免疫层析方法研究[J]. 中国农业科学, 2014, 47(19): 3883-3889.

LI Xiang-mei, WANG Zhan-hui, XIAO Xi-long, WANG Zhao-peng, WEN Kai, WU Xiao-ping, XIA Xi, WU Jin-xiao, JIANG Hai-yang. Development of a Colloidal Gold Immunochromatographic Technique for Simultaneous Detection of Quinolones and Gentamicin in Milk[J]. Scientia Agricultura Sinica, 2014, 47(19): 3883-3889.

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参考文献

[1] Li X M, Chen Y Q, Tang S S, He J K, Shang Y H, Xiao X L. Residue Depletion of Gentamicin in Swine Tissues after Intramuscular Administration. Journal of Agricultural and Food Chemistry, 2009, 57: 7356-7362.

[2] Zhu Y, Li L, Wang Z H, Chen Y Q, Shen J Z. Development of an immunochromatography strip for the rapid detection of 12 fluoroquinolones in chicken muscle and liver. Journal of Agricultural and Food Chemistry, 2008, 56: 5469-5474.

[3] 动物源食品中兽药最高残留限量. 农业部公告［2002］第235号，2002年12月24日.

The maximum residue limits of veterinary drugs in food products of animal origin. Bulletin of Ministry of Agriculture［2002］no. 235th, December 24, 2002.(in Chinese)

[4] Shaikh B, Allen E H. Overview of physical-chemical methods for determining aminoglycoside antibiotics in tissues and fluids of food producing animals. Journal of the AOAC International, 1985, 68: 1007-1013.

[5] Boison J O, MacNeil J D. Chemical analysis for antibiotics used in agriculture. Journal of the AOAC International, 1995, 4(6): 256-259.

[6] Haasnoot W, Stouten P, Cazemier G. Immnochemical detection of aminoglycosides in milk and kidney. Analyst, 1999, 124: 301-305.

[7] Loomans E E M G, Wiltenburg J V, Koets M. Neamin as an immunogen for the development of a generic ELISA detecting gentamicin, kanamycin, and neomycin in milk. Journal of Agricultural and Food Chemistry, 2003, 51: 587-593.

[8] Brown S A, Newkirk D R, Hunter R P. Extraction methods for quantitation of gentamicin residues from tissues using fluorescence polarization immunoassay. Journal of AOAC International, 1990, 73: 479-483.

[9] Bucknall S, Silverlight J, Coldham N, Thorne L, Jackman R. Antibodies to the quinolones and fluoroquinolones for the development of generic and specific immunoassays for detection of these residues in animal products. Food Additives and Contaminants, 2003, 20: 221-228.

[10] Huet A C, Charlier C, Tittlemier S A, Singh G, Benrejeb S, Delahaut P. Simultaneous determination of (fluoro) quinolone antibiotics in kidney, marine products, eggs, and muscle byenzyme-linked immunosorbent assay (ELISA). Journal of Agricultural and Food Chemistry, 2006, 54: 2822-2827.

[11] Bhogte C P, Patravale V B, Devarajan P V. Fluorodensitometric evaluation of gentamicin from plasma and urine by high-performance thin-layer chromatography. Journal of Chromatography B, 1997, 694: 443-447.

[12] Long Y H, Hernandez A, Kaale E. Determination of kanamycin in serum by solid-phase extraction, pre-capillary derivatization and capillary electrophoresis. Journal of Chromatography B, 2003, 784: 255-264.

[13] 尹茶, 吴玉田. 高效毛细管电泳法同时测定4种喹诺酮类药物. 药物分析杂志, 1997, 17(6): 371-373.

Yin C, Wu Y T. Determination of four quinolones by high performance capil lary electrophoresis. Chinese Journal of Pharmaceutical Analysis, 1997, 17(6): 371-373.(in Chinese)

[14] Liao L, Rao Y, Yang G X, Huang X H, Zeng Z L, Zeng Z L. Development of HPLC method for multiresidue determination of 12 quinolones in egg. Scientia Agricultura Sinica, 2008, 41(8): 2419-2424.

[15] Amound A I, Clark B J, Chrystyn H. Determination of gentamicin in urine samples after inhalation by reversed-phase high-performance liquid chromatography using pre-column derivatisation with o-phthalaldehyde. Journal of Chromatography B, 2002, 769: 89-95.

[16] Clarot I, Chaimbaul P, Hasdenteufe F. Determination of gentamicin sulfate and related compounds by high-performance liquid chromatography with evaporative light scattering detection. Journal of Chromatography A, 2004, 1031: 281-287.

[17] Preu M, Guyot D, Petz M D J. Development of a gas chromatography- mass spectrometry method for the analysis of aminoglycoside antibiotics using experimental design for the optimization of the derivatisation reactions. Journal of Chromatography A, 1998, 818: 95-108.

[18] Hatano K. Simultaneous determination of quinolones in foods by LC/MS/MS. Journal of the Food Hygienic Society of Japan, 2004, 45(5): 239-244.

[19] Cherlet M, Baere S D, Backer P D. Determination of gentamicin in swine and calf tissues by high-performace liquid chromatography combined with electrospray ionization mass spectrometry. Journal of Mass Spectrometry, 2000, 35: 1342-1350.

[20] Loffler D, Ternes T A. Analytical method for the determination of the aminoglycoside gentamicin in hospital wastewater via liquid chromatography- electrospray-tandem mass spectrometry. Journal of Chromatography A, 2003, 1000: 583-588.

[21] Zhou P, Lu Y, Zhu J, Hong J, Li B, Zhou J, Gong D, Montoya A. Nanocolloidal gold-based immunoassay for the detection of the N-methylcarbamate pesticide carbofuran. Journal of Agricultural and Food Chemistry, 2004, 52: 4355–4359.

[22] Jin Y, Jang J W, Han C H, Lee M H. Development of ELISA and immunochromatographic assay for the detection of gentamicin. Journal of Agricultural and Food Chemistry, 2005, 53: 7639-7643.