SN T 1604-2005 进出口动物源食品中氯霉素残留量的检验方法 酶联免疫法.pdf

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1、中华人民共和国出入境检验检疫行业标准SN/T 1604-2005 进出口动物源性食品中氯霉素残留量的检验方法2005-08-18发布酶联免疫法Inspection of chloramphenicol residues in animal derived food for import and export-Enzyme Iinked immunosorbent assay method 060608000129 2006-02-01实施中华人民共和国发布国家质量监督检验检茂总局SN/T 1604-2005 前言本标准的测定方法参照了德国r-biopharm公司生产的氯霉素酶联免疫定量测定试剂

2、盒的说明书。本标准的抽样和制样方法参照了欧盟98/179/EC指令，FAO/WHO农药残留法典委员会和德国商品检验机构所推荐的方法。本标准的附录A为资料性附录。本标准由国家认证认可监督管理委员会提出并归口。本标准起草单位:中华人民共和国上海出入境检验检疫局。本标准主要起草人:关蝶。本标准系首次发布的出入境检验检疫行业标准。I 1 范围进出口动物源性食品中氯霉素残留量的检验方法酶联免疫法SN/T 1604-2005 本标准规定了进出口动物源性食品中氯霉素残留量的抽样、制样和酶联免疫测定方法。本标准适用于进出口鸡肉、猪肉、虾仁和肠衣中氯霉素残留量的筛选检验，阳性结果须用其他方法进行确证。2 抽样和

3、制样2. 1 养殖场抽样将虾洗净，沿脊背剖开取肌肉组织10g50 g.总量500g. 2.2 屠宰加工厂抽样在屠宰线上，用清洁的锋利刀割取肌肉组织300g500 g. 2.3 肠衣集装箱抽样从一个集装箱第101桶至第250桶中随机抽取8桶，每桶抽取1副共计8副，约128个肠衣端头混合成一个样品进行检验。2.4 仓库抽样2.4. 1 检验批以不超过2500件为一检验批，同一检验批的商品应具有相同的特征，如包装、标记、产地、规格和等级等。2.4.2 抽样数量抽样数量见表1。襄1抽样数量批量/件最低抽样数/件125 1 26100 5 101250 10 251500 15 5011 000 17

4、1 0012 500 20 2.4.3 抽样方法按表1规定的抽样件数随机抽取，每件抽取一包，用清洁的锋利刀割取样品，每包不少于50g.总量不少于1峙。2.5 缩分小样肉类样品去除脂肪组织后用均质器均质;虾仁样品直接均质;肠衣样品用清洁的剪刀剪成长度不超过5mm的片段，当肠衣盐分较多时，先用重蒸锢水漂洗20min以去除盐分，沥干后再剪碎。每一样品都应分成至少两个相同的小样，每个小样的数量应能满足每次进行完整分析的用量。1 SN/T 1604一20052.6 样晶容器样品应装于合适的清洁干燥容器中，以保持样品的完整性和可追溯性。可采用聚乙烯塑料容器，玻璃容器等惰性材料容器(不允许使用橡胶容器)盛装

5、样品，然后再放入较大的干净容器中密封装运。2. 7 样晶封识应在每个样品容器的外表贴上标签，标签上注明样品名、样品编号、生产批号、抽样日期、抽样地点、堆位、抽样人员，并由抽样人员或官方人员加封。2.8 样晶保存抽样后样品应立即在一180C以下冷冻保存，OoC_50C条件下48h内送达实验室。在抽样和制样过程中，必须防止样品受到污染或发生残留物含量的变化。3 测定方法3. 1 测定原理本方法的测定基础是竞争性酶联免疫反应。氯霉素与氯霉素酶标记物竞争数量有限的氯霉素抗体，用酶标仪测量微孔榕液的吸光度值，氯霉素浓度与吸光度值成反比，按绘制的校正曲线定量计算。3.2 试剂和材料除另有规定外，所用化学试

6、剂均为分析纯，水为重蒸锢水。3.2. 1 乙酸乙醋。3.2.2 乙睛。3.2.3 正己烧。3.2.4 氯化铀溶液:0.5mol/L，经121C15 min高压灭菌。3.2.5 半对数坐标纸。3.2.6 氯毒素酶联免疫定量测定试剂盒。注z本标准3.5酶联免疫测定步骤中使用的试剂盒为德国r-biopharm公司产品，其他公司等效试剂盒的操作步骤可能略有不同，须经试验评估验证后使用。3.3 仪器和设备3.3. 1 酶标仪。3.3.2 均质器。3.3.3 离心机。3.3.4 氮气挥发器。3.3.5 振荡器。3.3.6 微量加样器:50L。3.3.7 微量多通道加样器:50L、100L。3.4 试样提取

7、和净化称取5.00g试样至离心管中，加入15mL乙睛-水溶液(84+16)，振荡1mn ,150C 3 000 r/min离心20mino移取3mL上清液至玻璃试管中，加入3mL乙酸乙醋和1mL氯化铀溶液，振荡1mn，静置分层后去掉下层的水相，有机相用氮气在600C水浴中吹干，残留物用1mL试剂盒提供的缓冲液溶解，加入1mL正己皖振荡1min, l 500 r/min离心10min.去掉上层的正己皖和脂肪层后供测定用。3.5 酶联免疫测定将一定数量的微孔条插入框架(标准液和样液分别做平行试验测定).记录标准液和样液的位置。加50L氯霉素酶标记物至每个微孔，加50L6个氯霉素标准液(0ng/L、

8、50ng/L、150ng/L、450 ng/L、1350 ng/L、4050 ng/L)和样液到各自的微孔，再加50L氯霉素抗体至每个微孔，轻拍混匀，用粘胶纸封住微孔以防榕液挥发.20oC-250C黑暗避光处孵育2h。倒掉微孔中的液体，洗板操作6次。加50L底物和50L发色剂至每个微孔中，轻拍混匀，20oC-250C黑暗避光处孵育30min。加SN/T 1604-2005 100L反应终止液至每个微孔中，轻拍混匀后，在10min内以空气为空白调零，测量并记录每个微孔溶液450nm波长的吸光度值。3.6 结果的计算和表述3.6.1 计算百分比眼光度值分别计算氯霉素标准液和样液的平均吸光度值，按式

9、(1)求得每个氯霉素标准液和样液的百分比吸光度值zA=三叫( 1 ) 式中zA一一-百分比吸光度值;S一一氯霉素标准液或样液的平均吸光度值;50一一一ong/L的氯霉素标准液的平均吸光度值。3.6.2 绘制校正曲线以百分比吸光度值(算术级)为纵坐标，以氯霉素浓度(ng/kg)(对数级)为横坐标，绘制出氯霉素标准液百分比吸光度值与氯霉素浓度的校正曲线(参见附录A)。每次试验均应重新绘制校正曲线。3.6.3 结果的襄述在3.6.2绘制的校正曲线上读取样液百分比吸光度值所对应的氯霉素浓度即为试样中的氯霉素残留量(ng/kg)。当测定值小于100ng/kg时，为氯霉素未检出;当测定值大于等于100ng

10、/同时，为氯霉素检测结果阳性，阳性结果须用其他方法进行确证。4 测定低限、回收率4. 1 测定低限本方法的测定低限为100ng/kg。4.2 回收率氯霉素添加浓度和回收率实验数据:100 ng/kg时，回收率为90.7 % 108. 6 % ; 1 000 ng/kg时，回收率为86.8%100. 6%; 2 500 ng/kg时，回收率为91.8%95. 3%。3 SN/T 1604-2005 附录(资料性附录)氯霉素校正曲线A 100 40 10 30 20 90 80 10 60 50 XVEa03Mq刨根鄙4050 1350 450 150 50 。氯霉素浓度/(ng/kg) 氯霉素校

11、正曲线固A.14 SN/T 1604-2005 Foreword The method of determination of this standard was drafted referring to the instruction manual of the enzyme immunoassakit for the quantitative analysis of chloramphenicol which made by r也iopharmCo. , Ltd. of Germany , through research , modification and verification.

12、The sampling and sample prep aration methods of this standard was drafted refering to the method which recommended bThe Council of the European Community Council Directive 98/179/EC, FAO/WHO Codex Committee on Pesticide residues and the commodities inspection institution of Germany. Annex A of this

13、standard is an informative annex. This standard was proposed by Certification and Accreditation Adiministration of the People s Re public of China. This standard was mainly drafted by Shanghai Entry-Exit Inspection and Quarantine Bureau of the Peoples Republic of China. This standard was mainly daft

14、ed by Guan Rong. This standard is professional standard of Entry-Exit inspection and quarantine promulgated for the first time. 5 SN/T 1604一20051 Scope Inspection of chloramphenicol residues in animal derived food for import and export二Enzyme linked immunosorbent assay method This standard specfes t

15、he methods of samplng , sample preparation and determinaton by ELlSA method of chloramphencol residues n animal derived food for mport and export. This standard s applcable to the screen determination of chloramphencol residues in chcken meat, pork,shrimp meat and sausage佣sngfor import and export. P

16、ositive results should be confirmed by another method. 2 Sampling and sample preparation 2. 1 Sampling in farm Cleaning the shrimp. Cutting the body open alone the spine to get the muscle 10g-50g, total weight is 500g. 2.2 Sampling in slaughter house On the slaught process , cutting out muscle 300 g

17、-500 9 with a cleaning sharp knife. 2. 3 Sampling in臼U臼gesingcontainer 8 tubs are taken at random from 101 th to 205 th tub n a contaner. Select one par from each tub to tal8阳r，about 128 termnals are mxed as one sample to detection. 2. 4 Sampling in warehouse 2. 4. 1 Inspection lot Each inspection l

18、ot of cargo should not exceed 2 500 packages. The characterstics of the cargo wth n the same nspecton lot, such as packing , mark, origin , specifcation and grade etc. , should be the same. 2. 4. 2 Quantity of回mpletaken The quantity of sample is taken accordng to table 1. 6 SN/T 1604-2005 Table 1 Qu

19、antity of sample Number of packages in Minimum number of pac阳geseach inspection lot to be taken 1-25 1 26-100 5 101-250 10 251-500 15 501-1 000 17 1001-2500 20 2.4.3 Sampling procedure A number of packages specified in table 1 are taken. Select one bag from each package , Cutting out samples at leas

20、t 50 9 from one bag with a cleaning sharp knife and total weight is not less than 1 kg. 2. 5 Sample portioning out Meat defated and homogenized thoroughly by blending in a high-speed blender; shrimp meat homoge nized directly; sausage臼singcut to a part with a clean scissors and the length could not

21、exceed 5 mm. When the sausage casing contain much salt , firstly rinse with double distilled water 20 min, cut to a part after water falling down. At least each sample should be divided into two equal por tions, the quantity of each portion should be sufficient for a whole analsis. 2. 6 Sample conta

22、iner AII samples should be placede in a clean and dry container to maintain the sample completeness and traceability. Samples can be placed in the inertia materials container which made of polyethylene plastic, glass etc. Cthe rubber container can not be used) , then change into a bigger clean conta

23、iner to seal up and transport. 2. 7 Sample sealing and labeling The label should be ladeled outside of each sample container. The label indicates the name of sample, the serial number of sample , the production batch number, the date of sampling , the sampling loca tion , the stack position , the sa

24、mpling person , and sealed by the sampling person or official man. 2. 8 Storage of sample After sampling , the test sample should be stored below - 18.C immediately, and deliver to the labo ratory at OC -5C within 48 h. In the course of sampling and sample preparation , precaution must be taken to a

25、void contamination or any factors which may cause the change of residue content. 3 Method of determination 3. 1 Principle of determination 7 SN/T 1604-2005 The basis of the test is the competitive enzyme-linked immuno reaction. Chloramphenicol and chlor amphenicol enzyme conjugate compete for the li

26、mited anti-chloramphenicol antibod. The absor bance value of each well solution is measured by microwell system. The chloramphenicol concentra tion is inversely proportional to the absorption value, and compare with the calibration curve for quantative measurement. 3. 2 Reagents and materials In thi

27、s standard ,all the chemical reagents shoud be analtically pure , water is double distilled wa ter. 3.2.1 EthI acetate. 3.2.2 Acetonitrile. 3. 2. 3 n-hexane. 3.2.4 Sodium choride solution: 0.5 mol/L autoclave at 121 C for 15 min. 3.2.5 Semi-logarJthmic graph paper. 3. 2. 6 Enzyme immunoassakit for t

28、he quantitative analysis of chloramphenicol. Note: The r-bio阶armkit of Germany is used in this standard 3. 5 ELlSA determination, the testing process of an other equal kits maybe a little differ，回itshould be used after testing , evaluation and verification. 3.3 Ap阴阳tusand町uipment3. 3. 1 Microwell sy

29、stem. 3.3.2 Homogenizer. 3. 3. 3 Centrifuge. 3. 3. 4 Nitrogen Evaporator. 3. 3. 5 Vibrator. 3.3.6 Pipettes:50L 3.3.7 Multichannel pipette:50L，100L 3. 4 Sample extraction and cleanup Weith 5. 00 9 of the test sample into a centrifuge tube, add 15 mL of acetonitrile-water mixture (84+ 16) , vibrate 1

30、min. Centrifuge at 150C 3000 r/min for 20 min. Remove 3 mL of supernatant in-8 SN/T 1604-2005 to a glass centrifuge tube , add 3 mL eth1 acetateand and 1 mL sodium choride solution , vibrate 1 min,let stand to separate,discard the lower aqueous phase , the organic phase is blew off with ni trogen in

31、 600C water bath. Dissolve the residue in 1 mL of buffer from kit ,add 1mL n-hexane , and vi brate 1min, centrifuge 1 500 r/min for 10 min , after discarding the upper n-hexaneand and fat laer completel，use the lower aqueous phase in the ELlSA assay. 3. 5 ELISA determination Insert a sufficient amou

32、nt of microtiter wells into the microwell holder for all standards and samples to be run in duplicate. Record standard and sample positions. Add 50L of diluted chloramphenicol enzyme conjugate to the bottom of each well and add 50L of six kinds of prepared chloramphenicol standard solution (0 ng/L,5

33、0 ng/L,150 ng/L,450 ng/L, 1 350 ng/L,4 050 ng/L) and sample solution to separate duplicate wells. Add 50L of diluted anti-chloramphenicol antibodsolution to each well , flap lightly and mix thoroughly, sealing all wells with adhesive paper to avoid solution evapora ting , incubate for 2 h at 20 C -2

34、5C in darkness. Pour the liquid out of the wells and wash the wells six times. Add 50L of substrate and 50L of chromogen to each well , flap lightly and mix thor 。ughlyandincubate for 30 min at 20 C -25C in darkness. Add 100L of stop solution to each well, flap lightly and mix thoroughl. To use atmo

35、sphere as blank zero , measure and record the absor bance value of each well solution at 450 nm within 10 min. 3. 6 Calculating the result and express 3. 6. 1 Calculating absorbance value in percent啕esCalculate the mean absorbance value of each chloramphenicol standard and sample solution individu a

36、ll， the chloramphenicol standard and sample absorbance value are calculated in percentages accord ing to the formula (1): A=去x100% . ( 1 ) where: A-absorbance value in percentages; 5-一一chloramphenicolstandard or sample solution; 50-一-meanabsorbance value of 0 ng/L concentration of chloramphenicol st

37、andard solution. 3.6.2 Plot臼librationcurve Make the absorbance value in percentages (arithmetic grade) as ordinate, and chloramphenicol con centration (ng/kg) (Iogarithmic grade) as abscissa , plot the calibration curve of the absorbance val ue in percentages and chloramphenicol standard concentrati

38、on (see Annex A). A new calibration curve should be prepared for each determination. 3. 6. 3 Calculating the result The chloramphenicol concentration of the test sample solution can be read corresponding to the per centage absorbance value of each sample from 3.6.2 calibration curve and namely equal

39、 to the resi due content of chloramphenicol in the test sample (ng/kg). The chloramphenicol residues is not de tected when the result is 100 ng/kg; The chloramphenicol residues is positive when the result is 9 SN/T 1604-2005 二三100ng/kg. Positive results should be confirmed by another method. 4 limit

40、 of determination and recovery 4. 1 Limit of determination The limit of determination of this method is 100 ng/kg. 4. 2 Recovery The experimental data of this fortifying concentrations of chloramphenicol and recoveries are as fol lows, At 100 ng/kg, the recovery is 90.7%.108.6%; At 1 000 ng/炮，the re

41、covery is 86.8%-100.6%; At 2 500 ng/阔，the recovery is 91.8%-95.3%. 10 SN/T 1604-2005 Annex A (Informative Annex) Chloramphenicol calibration curve 100 90 80 70 60 40 30 50 20 10 次)Egg-TBgfsAe4050 1350 450 150 50 。Chloramphenicol concentration / Cng/kg) Chloramphenicol calibration curve Fig A. 1 的CON-gFFZm中华人民共和国出入境检验检疫行业标准进出口动物源性食晶中氯霉素残留量的检验方法酶联免瘟法SN/T 1604-2005 唔中国标准出版社出版发行北京复兴门外三里河北街16号邮政编码:100045 网址电话:6852394668517548 中国标准出版社秦皇岛印刷厂印刷晤印张1字数21千字2005年11月第一次印刷开本880X 1230 1/16 2005年11月第一版峰定价10.00元如有印装差错由本社发行中心调换版权专有侵权必究举报电话:(010)68533533书号:155066 2-16448 1604-2005