1、5 中华人民共和国出入境检验检疫行业标准SN/T 1966一2007水产品中氯霉素残留检测方法放射受体分析法Determination of chloramphenicol residues in aquatic product Radio寸eceptorassay method 2007-08-06发布2008-03-01实施中华人民共和国发布国家质量监督检验检痊总局SN/T 1966-2007 前言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s中华人民共和国广东出入境检验检疫局。本标准主要起草人z相大鹏、陈小雪、张林田、陈燕勤、梁希扬、罗惠明、林文华、庄逸林。本标准系首次发
2、布的出人挠检验检疫行业标准。I 1 范固水产品中氯霉素残留检测方法放射受体分析法SN/T 1966-2007 本标准规定了水产品中氯霉素残留量测定的制样和放射受体分析(Charm11 )方法。本标准适用于水产品中氯霉素残留量的筛选测定。2 规范性引用文件下列文件中的条款通过本标准的引用而成为本标准的条款。凡是注日期的引用文件，其随后所有的修改单(不包括勘误的内容)或修订版均不适用于本标准，然而，鼓励根据本标准达成协议的各方研究是否可使用这些文件的最新版本。凡是不注日期的引用文件，其最新版本适用于本标准。GB/T 6682 分析实验室用水规格和试验方法3 试样制备和保存3. 1 试样的制备从全部
3、祥品中取出不少于500g有代表性试样，用高速组织捣碎机捣碎均匀，将捣碎的样品充分混匀。装入清洁的容器肉，加封并做标识。在制样的过程中，应防止样品受到污染或发生残留物含量的变化。用于测定的样品细菌数不能超过10徊。3.2 试样保存将试样于一18C以下冷冻保存.4 B固定方法4. 1 原理测定的基础是竞争性受体免疫反应。样品中的氮霉素残留经提取后，与HJ标记的氯霉素竞争细菌细胞中的氯霉素特异性受体，样品中的氯霉素药物含量越高，竞争结合的位点越多，HJ标记的氯霉素结合的位点则越少。在一定温度反应后，离心分离，清除未结合的HJ标记的氯霉素，加闪烁液后用液体闪烁计数仪测定样品中的HJ含量cpm值(cou
4、ntper minute，即每分钟脉冲数).cpm值越低，则样品中的氯霉素残留越高。4.2 试剂和材料4.2. 1水GB/T 6682规定的一级水。4.2.2 组织中氯霉素检测Charm11试剂盒每批新试剂盒使用前须进行性能监测。4.2.2. 1 竞争性结合试如1:应贮存在15C或以下，在室温(l5C25C)下最多放置6h. 4.2.2.2 HJ标记的氯霉素应贮存在15C或以下，在室温(l5C25C)下最多放置6h. 1) Charm II竞争性受体结合组织氯霉素测试试剂盒是由larm公司提供的产品的商品名称。给出这一信息是为了方便本标准的使用者，并不表示对该产品的认可。如果其他等效产品具有相
5、同的效果，则可使用这些等效产品.SN/T 1966-2007 4.2.2.3 阴性对照浓缩溶液z干粉贮存于2C6C.使用时取浓缩干粉按标签上标记配制成阴性对照浓缩溶液，配制好的溶液可在2C6C冰箱或冰浴中保存48h;于一15C以下的冰箱中，则可保存2个月，佼用时将其解冻，解冻后的溶液在2C6C冰箱或冰浴中保存24人4.2.2.4 10g/L氯霉素标准溶液=于fJ贮存于2C6C.使用时按照标签溶解，氯霉素的浓度为10问/L.溶液的保存方法同4.2. 2. 2所述。4.2.2.5 萃取缓冲溶液=干粉贮存于ZC6C使用时按照标签溶解，该缓冲溶液可在2C6C下保存2个月。4.2.2.6 M2缓冲液z干
6、粉贮存于2C6C.使用时按照标签溶解，该缓冲溶液可在2C6C下保存2个月。4.2.2.7 闪烁液。4.3 仪器和设备4.3. 1 液体闪烁汁数仪(LSC) 4.3.2 旋涡振荡器。4.3.3 恒温孵育器。4.3.4 离心机，最大转速为4000r/min. 4.3.5 高速组织捣碎机.4.3.6 硅棚酸盐玻璃试管及试管塞。4.4 分析步骤4.4. 1 阴性和阳性对照涩的配制4.4. 1. 1 阴性对照法的配幸IJ取2mL阴性对照浓缩溶液也止血且毕制成阴性对照液，该对照液可在室混05C25C)下3 300 r/min离心10min。4.4.2.3 吸出上消液，注意不要将漂浮的脂肪颗粒混入上消液内。
7、用pH试纸检查提取溶液pH值是否为7.58. O.若偏离，用M2缓冲液(4.2.2.6)或1mol/L盐R空调节pH至7.58. O.即为样品提取液。4.4.3 测定4.4.3. 1 用压杆将竞争性结合试jlJ压入一洁净的硅砌酸盐玻躏试管内，加入300L水，用涡旋振荡器振荡10s，使试剂振碎完全。4.4.3.2 加5.0mL样品提取液(4.4.2.3).或阴性对照液(4.4.1.1)或阳性对照液(4.4.1.2)到试管中，混匀15s，使样品上下混匀15次，置于恒温孵育器中于50C土1C孵化3min，取出混匀15s，再置于恒温孵育器中50C士1C孵化3mino 4.4.3.3 用压杆将HJ标记的
8、氮霉素压入试管中，用旋涡混合器振荡15s，使样品上下混合15次，置于恒温孵育器中50C土1C孵化3min ,. SN/T 1966-2007 4.4.3.4 取出试管，于3300r/min离心10min，立即取出试管.倒掉上清液，用棉签将试管边缘的污溃擦去。4.4.3.5 加300L水到试管内，振荡并混合i昆匀，再加入3.0mL闪烁液(4.2.2.7)至试管中，涡旋混匀至试管内没有不均的云状物。4.4.3.6 将试管放人液体闪烁计数仪内，在3HJ频道进行60s LSC计数，读cpm值。4.5 控制点的确定控制点是判断样品阴性与初筛阳性的一个界定值，可根据筛选水平自行设定。对于同一批号的试剂，正
9、常情况下只需测定1次控制点。控制点设定步骤如下2称取20.0g均质好的空白组织样品，加入0.6mL 10问/L氯霉素抗生素标准溶液，按4.4.2试样提取和4.4.3放射免疫测定z测定六个加标样品的cpm值，求出cpm平均值乘上系数1.2，即为筛选水平0.3g/kg的控制点。注空白样品应是不吉抗生素残回tJ样占fl，_-NP如果在11导某个样品cpm但在剧性对照液的cpm值土20%左右波动，可认为其不古氯霉素抗生素。IJ 5 结果判定I.-.中户可/ ;.,0/ 5. 1 将测得结果与控制点对照，当样品cpm倍死于控制点50_.以上时，判定样品为阴性飞&P样品中氯./ ./ 霉素含量低于筛选水平
10、。/ 5.2将测得结果与控制点对照，当ril11轩划、于控制点，视为咱筛怀疑阳性，此时应重新测定样品、阴性对照液和阳性对照曹贯的pmr(, Ift性计何?贸啪曹对ff液的cpm但需在正常范围波动见试iflj盒性能监割) ，当重新测定样的cpmi在大于控制点，判定为?阴性;小于或等于控制点，则判定为初筛阳性0 I仁川;二!5.3 将世H导结果与控制点对照，g(;挥斗目布古1自1盖面苍白IEZ77且不足50时，重新计数，如果重新计数7 检测低限在水产品巾，氯霉素残留检测低限为O.3g/kg。8 确证如被测样品中氯霉素残留筛选结果为阳性的，应用其他方法进行确证。3 SNjT 1966-2007 Fo
11、reword This standard was proposed byand is under the charged of certification and accreditation administra tion of the People s Republ ic of China. This standard was drafted by Shantou Entry-Exit Inspection and Ouarantine Bureau This standard was mainly drafted by Da-peng Xiang ,Xiao-xue Chen ,Un-ti
12、an Zhang , Yan-qin Chen ,Xi yang Uang , Hui-ming Luo , Wen-hua Un, Yi-lin Zhua吨，This standard is a professional standard for entry-exit inspection and quarantine promulgated for the first time. 4 SN/T 1966-2007 Determination of chloram阶enicolresidues in aquatic pr创uct一Radio-receptor assay method 1 S
13、cope This standard specifies the methods for the sample preparation and the determination of chloram phenicol residues by Charm 1I in aquatic products This standard is applicable to the screening determination of chloramphenicol residues in aquatic products. 2 Normative references The following norm
14、ative documents contain provisions which. through reference in this text. con stitute provisions of this standard. For dated references. subsequent amendments to. or revisions of. any of these publications do not apply. However. parties to agreements based on this standard are encouraged to investig
15、ate the possibility of applying the most recent editions of the normative documents indicated below. For undated references. the latest edition of the normative documents referred to appl ies. GB/T 66S2 Water for analytical laboratory use-Specification and test methods 3 Preparation and storage of s
16、ample 3. 1 Preparation of sample Take the representative portions from the whole primary sample for not less than 500 9 and blend in a high speed blender. Homogenize thoroughly. Keep the prepared sample into a clean bottle. seal and label In the Course of sample preparation. precaution should be tak
17、en to avoid contamination or any fac tors that may cause the change of residues content. Do not use samples with bacterial counts excess 10 /g. 3. 2 Storage of sample Store sample at below -1St. 5 SN/T 1966-2007 4 Method of determination 4. 1 Principle of detennination The determination is based on
18、a competitive receptor immunoassay. The chloran、phenicolthat ex tracted from the sample compete for the binding sites of the specific receptor, which attached to a microbial cell , with an exempt amount of HJ labeled chloramphenicol. The higher of chlorampheni col residues in the sample , the more r
19、eceptor sites they competitively bind , while the bound HJ la beled chloramphenic咀1are less. After the reaction at specified temperature , centrifuge and discard the unbound HJ labeled chloramphenicol. Then add the scintillation fluid and determine by a liquid scintillationunter for the cpm fcnt per
20、 minuteyfth旧inIthe sample e现tract.The lower 工专-d气-.: cpm is , the higher chloramphenicrsij;s there isii it sample. 4.2. 1 4.2.2 二J 旷吨主;手王气二For each new 101 of kits,performal:mon1toringmust bedone before used 4.2. 2. 1 The binding reagent, Store 气回nbe kept at room temperature (15 C -25C) for up to 6
21、h. 4.2.2.4 10g/L chloramphenicol standard,Store dry standard at 2C -6C. Reconstitute as direct ed on the label. Shake well. The concentration of the reconstituted standard solution is 10g/L. The conserved method is same as 4.2.2.2. 4.2.2.5 Extraction buffer,Store dry buffer at 2C -6C. Reconstitute b
22、uffers as directed on the la bel. Reconstituted buffers may be held at 2C -6C for up to 2 months. 1) Charrn n Test kit For chloramphenicol in Tissue is the commercial kit provided by Charrn Corporation. This informa tion is given for the convenience of the users of this standards , not meaning the c
23、ertification of this product. Other similar proclucts may be used for this standard. if they have the 5ame effect. 6 SN/T 1966-2007 4.2.2.6 M2 buffer,Store dry buffer at 2C -6C. Reconstitute buffers as directed on the label. Re constituted buffers may be held at 2C -6C for up to 2 months. 4.2.2.7 Sc
24、intillation fluid. 4.3 Apparatus and equipment 4.3. 1 Liquid scintillation counter. 4.3.2 Vortex mixer. 4.3.3 Inculcator, Thermostaticny controlled. 4.3.4 Centrifuge,Capable to 4 000 r/min 4.3.5 High-speed blender. 4.3.6 Borosilicate glass test t 4.4 Analysis procedure Add 0.3 mL of 10g/L chloramphe
25、nicol standard (4.2.2.的to6 mL performance negative concen trate. Mix well. Dilute 2 mL of this mixture with 6 mL extraction buffer. It can be kept at room tem perature (15C -25C) for up to 6 h. Used for performance monitoring for instruments and reagents and the determination of control point 4. 4.
26、2 Sample extraction 4.4.2. 1 Weigh 20.0 9 of the homogenized sample to a 50 mL centrifuge tube. Add extraction buff er (4.2.2.5) up to the 40 mL mark. Vortex for 5 min. 4.4.2.2 Incubate in 80 C :t2C for 45 min. Then place centrifuge tube in ice water bath for 10 min. 7 SN/T 1966-2007 Centrifuge for
27、10 min at 3 300 r/min. 4.4.2.3 Pipet top layer.avoid decanting any fat particles with supernatant. Check pH with indicator strips. If pH is departure.adjust the pH with M2 Buffer (4.2.2.6) or 1 mol/L HCI (dropwise) to 7.5-8. O. The sample extract is ready for determination. 4. 4. 3 Determination 4.4
28、.3. 1 Using the blunt end of a pen or pencil. push the bindi咱reagenttablet into a clean borosilicate glass test tube. Add 300 ,.,L of water to the tablet. Vortex for 10 s to break up the tablet. 4. 4. 3. 2 Add 5. 0 mL sample extract (4. 4. 2. 3) or negative control (4. 4. 1. 1) or positive control (
29、4.4. 1. 2) to the test tube. Vortex for 15 s.causing sample to rise and fall 15 times. Place the tube in incubator at 50(;:!: 1(; for 3 min. Remove the tube from incubator. Mix for 15 s and place the tube in incubator at 50(; :!: 1 (; for 3 min again. 4.4. 3. 3 Using the blunt end of a pen or pencil
30、. push the HJ antimicrobial tablet into the test tube. Vortex for 15 s.causing sample to rise and fall15 times. Place the tube in incubator at 50(;:!: 1(; for 3 min. 4.4.3.4 Remove the tube from incubator and centrifuge at 3 300 r/min for 10 min. Pour off extract and blot edge of tube on absorbent t
31、owel immediately. 4.4.3.5 Add 300L of water and vortex to mix well. Add 3.0 mL scintillation fluid (4.2.2.7) to the test tube. vortex till there is no inhomogeneous cloudy sediment in the tube. 4.4.3.6 Count in Liquid scintillation counter for 60 s. Read cpm (count per minute) on HJ chan nel. 4.5 De
32、termination of Control Point The control p旧intis the cut-off value between negative and presumptive positive results. which can be defined according to the screening level. A new control point should be established for each new batch of reagents. Determinate the control point as the procedure below,
33、 Weigh 20. 0 9 of the homogenized blank sample.spike 0.6 mL of 10g/L chloramphenicol standard solution. Extract this spiked sample as the procedure described in 4. 4. 2 and run radio-receptor assay as specified in 4.4.3. Determine cpm of the 6 spike samples. Average cpm results and multiply by a fac
34、tor 1. 2. And it is the control point of O. 3g/kg screening level. Note, The blank回mpleshould be free of chlorampheniI.which c田nis flu口uati咱within:t 20 % of the n咱ativentrol8 S:J jT 1966-2007 5 Determinant of results 5. 1 If the sample cpm is greater than the control point by 50 points or above. the
35、 sample is nega tive. which chloramphenicol content is less than the screening level. 5.2 If the sample cpm is less than or equal to the control point. the sample is presumptive posi tive. The sample should be retested along with a negative control and positive control. The cpm of the negative contr
36、ol and the positive control must be fluctuating between the expected range. (See performance monitoring of the kiO. If the retest cpm is greater than the control point. the sample is negative; otherwise,the sample is screening positive, 5. 3 If the sample c口mis greater than the control point within
37、50 points. the sample should be re counted. If the recount is less than the control point. the sample is screening positive. 6 Performance monitoring of the kit For each new lot of reagents. establish a zero control average by running 6 negative controls and av eraging the results. When testing pres
38、umptive positives. test a negative control and positive con trol. compare the cpms to which of the zero control average to verify the reagents and equipment are working properly a) The negative control should test within :!: 15% of the zero control average, b) The positive control should test less t
39、han the control point, c) The ratio of positive control cpm and negative control cpm should be less than 0.7, d) If the results are not met the conditions above. the zero control average and the control point should be determined again .and the sample should be retest. 7 Limit of determination The l
40、imit of determination of this method for chlorampheniI residues in aquatic pr，ucts is O. 3阅jkg.8 Confirmation test If sample is presumptive positive. it should be confirmed with confirmation method 9 中华人民共和国出人挽检验检疫行业标准水产品中氯霉素残留检测方法放射受体分析法SN/T 1966一2007争峰中国标准出版社出版北京复兴门外三旦河北街16号邮政编码，100045网址电话2日852394668517548 中国标准出版社秦皇岛印刷厂印刷开丰880X12301/16 印张1字数18千字2007年11月第一版2007年11月第一次印刷印数1-2000电h书号，155066.2-18247 定价10.00元
