【作者】 宋士波；

【导师】 袁宗辉；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2002， 硕士

【摘要】 为监控动物性食品中抗菌药物残留，保障人民健康，本实验研究微生物法快速筛选试剂盒的开发与应用，即用于猪肾组织中抗菌药物残留检测的在场拭子法试剂盒(Swab Test On Premises，STOP)，抗菌药物快速筛选试剂盒(Fast Antimicrobial Screening Test，FAST)和用于猪活体尿液中抗菌药物残留筛选的活体动物拭子法(Live Animal Swab Test，LAST)。FAST试剂盒培养基pH为7.3，接种巨大芽孢杆菌。本实验中研发的STOP和LAST试剂盒培养基相同，pH为7.3，接种枯草芽孢杆菌。 本实验中用添加法测定了STOP和LAST试剂盒对青霉素、氨苄青霉素(β-内酰胺类)，金霉素(四环素类)，新霉素、庆大霉素(氨基糖甙类)，恩诺沙星(喹诺酮类)以及红霉素(大环内酯类)等不同种类抗菌药物的检测限。结果表明，对于以上7种常用的抗菌药物，STOP和LAST试剂盒的检测限不超过我国规定的最高残留限量。 实验中用添加法测定了STOP和LAST试剂盒对以上7种抗菌药物(每种药物3个浓度。包含最低检测限)的回收率及变异系数。STOP试剂盒回收率范围：青霉素51.5％～95.7％、氨苄青霉素68.5％～102.3％、红霉素51.4％～82.3％、金霉素52.7％～88.9％、庆大霉素51.4％～87.3％、新霉素50.5％～89.0％、恩诺沙星46.1％～79.4％。批间变异系数均小于15％，批内变异系数不超过10.1％。LAST试剂盒的回收率范围：青霉素41.4％～90.5％、氨苄青霉素77.6％～107.3％、红霉素79.8％～124.7％、庆大霉素65.9％～118.8％、新霉素73.6％～107.7％、金霉素42.2％～61.4％、恩诺沙星53.1％～99.6％。回收率的批间变异系数均小于15％，批内变异系数不超过14.2％。 在最高残留限量浓度，分别测定了STOP、LAST、和FAST试剂盒对以上7种药物的假阴性，其假阴性率分别为：STOP试剂盒，青霉素4.3％、氨苄青霉素4.0％，其他5种抗菌药物没有发现假阴性。LAST的试剂盒，青霉素7％、氨苄青霉素4％，其他5种抗菌药物没有发现假阴性。FAST试剂盒，青霉素12％，其他6种抗菌药物没有发现假阴性。 对于10种常用的抗菌药物(包括磺胺二甲嘧啶、氯霉素、林可霉素和三甲氧苄胺嘧啶，组织中每种药物添加3个浓度)，STOP、LAST、FAST试剂盒同国外同种产品做了比较，结果表明三种试剂盒达到或接近国外同种产品的检测水平。 2002年5～6月，用STOP试剂盒抽样分析了武汉市市售动物性食品(猪肾105份、猪肝100份、肌肉100份)中抗菌药物残留的情况。结果发现，45份肝组织、14份肾组织、1份肌肉中出现阳性结果，说明当前的残留问题不容乐观。试剂盒的稳定性(菌液、培养基、纸片)考察表明，三种试剂盒中应用芽孢悬液以及STOP检测用培养基至少2个月内可以保持有效，新霉素阳性对照纸片9个月来效价没有显著变化。 本实验研究填补了我国抗菌药物残留微生物筛选方法的空白，具有重要的学术意义和应用价值。本实验研究的三种试剂盒(STOP、LAST、FAST)作为国内首次开发的抗菌药物残留微生物快速检测工具，具有应用面广、操作简便、性能稳定、成本低廉等诸多优点，将会广泛适用于检疫部门对动物性食品中抗菌药物残留的日常检测、进出口检疫以及养殖场、屠宰场、肉联厂、食品加工厂进行的在场残留检测工作，具有较大的市场开发价值和应用前景。更多还原

【Abstract】 The studies on Swab Test on Premises (STOP), Fast Antimicrobial Swab Test (FAST) and Live Animal Swab Test (LAST) test kits were carried out for the detection of antimicrobial residues in swine tissues and urine. The culture media of STOP and LAST test kits was inoculated with Bacillus subtilis with pH7.3, while the culture media of FAST test kit was inoculatd with Bacillus megaterium with pH7.3. STOP and FAST test kits screened swine kidneys, and LAST test kit screened swine urine for antimicrobial residues.The limits of detection of STOP and LAST test kits for 7 antimicrobial drugs (Penicillin, Ampicillin. Erythromycin, Chlortetracycline, Gentamycin, Neomycin, Enrofloxacin) were determined in swine kidney and urine, which were not above the MRLs established in China. The recovery and its coefficients of variation (CV), of the 7 antimicrobial drugs from tissue and urine, were determined by using the STOP and LAST test kits respectively. The range of recovery of STOP test kit was shown as followed: Penicillin 51.5%~95.7%, Ampicillin 68.5%~102.3%. Erythromycin 51.4%~82.3%, Chlortetracycline 52.7%~88.9%, Gentamycin 51.4%~87.3%. Neomycin 50.5%~89.0%. Enrofloxacin 46.1%~79.4%. The interday CV was below 10.1%, and the intraday CV was below 15%. The range of recovery of LAST test kit was shown as followed: Penicillin 41.4%~90.5%, Ampicillin 77.6%~107.3%, Erythromycin 79.8%~124.7%. Chlortetracycline 42.2%~61.4%, Gentamycin 65.9%~118.8%, Neomycin 73.6%~107.7%, Enrofloxacin 53.1%~99.6%. The interday CV was below 14.2%, the intraday CV was below 15%.It was found that the false proportions of FAST. STOP. LAST test kits to the 7 drugs were very low: STOP test kit. Penicillin 4.3%. Ampicillin 4.0%. the others 0%; LAST test kit. Penicillin 7%, Ampicillin 4%, the others 0%: FAST test kit. Penicillin 12%. the others 0%.A comparison was made between the three test kits and the same kind foreign test kits, which gave quite similar results to 10 antimicrobial drugs (including Lincomycin, Chloromycetin. Sulfadimidine. Trimethoprim. Olaquindox). It could be concluded that the that test kits attained or neared to the level of abroad products.The antimicrobial residues in 105 kidney. 100 liver and 100 muscle samples of swine, wenh were collected from retail outlets in Wuhan between May and June 2002. were in tigated by using STOP test kit. Positive STOP results were found in 14 kidney, 45 liver and ! muscle. In the end, the stability of test kits was discussed in this paper. The spore suspentions and assay culture of STOP test kit were valid for at least 2 months, and the N5 disc was steady for at least 8 months.It was concluded that the three test kits, as the first microbiological screening tools developed in China for the detection of antimicrobial drugs residues in swine tissue and urine, were suit to be used by authorities in official quarantine procedures for the antimicrobial residues control, or as in-plant methods in slaughterhouse, meat-packing plant or stockyard to screen for residues of antimicrobial drugs. The microbiological screening methods were practical, steady, inexpensive and easy to perform, which should have enormous promise in our country.更多还原