1、ls己中华人民共和国出入境检验检疫行业标准SN /T 1134-2002 进出口肉及肉制品中维吉尼毒素残留量检验方法杯碟法Method for the determination of virginiarnycin residues in meats and meat products for import and export -Cylinder method 2002- 08-02发布2003 -0个01实施中华人民共和国发布国家质量监督检验检疫总局SN /T 1134-2002 前言本标准中的测定方法是采用日本厚生省1990年所编制的畜、水产食品中的残留物质检验方法中维吉尼霉素检验方法，
2、其技术内容与原方法相同.具体操作方法稍加改动，经试验和验证后，按规定要求作了编辑性修改。本标准中同时制定了抽样和制样方法。测定低限是根据国际上对肉品中维吉尼霉素残留量和测定方法灵敏度而制定的。本标准的附录A是规范性附录，附录B是资料性附录。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:中华人民共和国天津出入境检验检疫局。本标准主要起草人:王素琴、何霖、孙俐、陈子红。本标准系首次发布的出入境检验检疫行业标准。1 范围进出口肉及肉制品中维吉尼霉素残留量检验方法杯碟法SN/T 1134二2002本标准规定了进出口肉及肉制品中维吉尼霉素残留量检验的抽样、制样和杯碟测定方法。本标准适用于
3、进出口冻鸡肉中维吉尼霉素残留量的检验。2 抽样和制样2. 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相同的特征，如包装、标记、产地、规格和等级等。2.2 抽样数量批量/件最低抽样数/件125 1 26100 5 101 250 10 251 500 15 5011 000 17 1 001 2 500 20 2. 3 抽样方法按2.2规定的抽样件数随机抽取，逐渐开启。每件至少一袋为原始样品。如每件中无小包装或有小包装，但每袋重量超过2kg，则可用灭菌刀从每件中割取不少于100g样品，混合后置于清洁容器内作为混合样品。原始样的总量不少2峙，加封后标明标记，及时送交实验室。2.4
4、 试样制备从原始样品中取出部分有代表性的样品，将可食部分用绞碎机绞碎.充分混匀，用四分法缩分至不少于500g，作为试样，装入清洁容器内，密封后，标明标记。2. 5 试样保存将试样于18C以下冷冻保存。注:在抽样和制样的操作过程中，必须防止样品受到污染后发生残留物含量的变化。3 测定方法3- 1 方法提要用乙醇提取试样中残留的维吉尼霉素，经均质、离心后取上清液作为样液，取上清液与结膜干燥棒杆南作用，根据产生抑菌圈直径大小，并查阅标准曲线，计算出样品中维吉尼霉素的残留量。SN/T 1134-2002 3- 2 试剂和材料除另有规定外，所用试剂均为分析纯，水为蒸馆水。本方法标准品及其标准珞液均以效价
5、计。3.2.1 无水乙醇。3- 2.2 元水甲醇。3. 2.3 甲醇十水(1十2)。3. 2. 4 生理盐水:称取8.5g氧化饷，容于1000 mL水中，121C灭菌15min。3. 2. 5 磷酸二氢押。3. 2.6 磷酸氢二铀。3. 2.7 磷酸盐缓冲液CpH6.0):称取3.5g磷酸二氢押(精确至0.1g)搭于400mL水中，另外称取3.0 g磷酸氢二铀(精确至0.1g)，洛于400mL水中，将两种培液混合在一起定容至1000 mL。3. 2. 8 维吉尼霉素标准品。3. 2.9 维吉尼霉素标准贮备液:准确称取适量的维吉尼霉素标准品，用甲醇溶液(3.2.3)溶解后定容至50 mL，配成1
6、000 吨/mL的维吉尼霉素标准贮备液，于4C保存，一周内使用。3- 2.10 维吉尼霉素标准工作液:吸取一定量的标准贮备液，用磷酸盐缓冲液分别稀释成浓度为0.025g/mL、0.05f-Lg/mL、O.1g/mL、O.2g/mL、0.4g/mL和O.8g/mL标准工作溶液，以上稀释均须当日配制。3. 2. 11 实验菌种:结膜干燥棒杆菌CCorynebacteriumxerosis)。3. 2. 12 培养基1(见附录Al)。3. 2. 13 培养基II(见附录A2)。3. 2. 14 培养基(见附录A3)。3. 3 仪器和设备3. 3. 1 游标卡尺:测量范围omm200 mm，精度0.0
7、2mm，或抑菌圈测量仪。3. 3. 2 牛津杯:高10mm:!:O. 1 mm，内径6.0mm士0.1mm，外径8.0mm:!:O. 1 mm。3.3. 3 离心机:不低于3000 r/min。3-3- 4 恒温培养箱:30C士1C , 36 C士1C , 50 C士1C，隔板保持水平。3- 3. 5 高压灭菌器。3.3. 6 绞碎机z不低于3000 r/min。3.3.7 均质器:不低于10000 r /min，带均质杯250mL。3- 3. 8 培养皿:内径90mm，底部平整光滑的玻璃皿或塑料皿，并应配有陶瓦盖。3.3.9 其他:玻璃板、吸管、毛细管等。3.4 测定步骤3. 4.1 试液制
8、备准确称取10g试样(精确至0.1g)于均质杯中，加入10mL乙醇，均质2min后移入离心管中，以3000 r/mi口离心20min，取其上清液，作为试液。3. 4.2 菌蔽的制备将实验菌种(3.2.11)安瓶瓶的上部消毒后敲碎.加入少量培养基E于试验菌种中，使其充分溶解并移至盛有培养基E的试管中?昆均，置37C土1C培养24h，将培养物划线接种到培养基I斜面上。用适量灭菌生理盐水冲洗菌苔并转入灭菌试管中，4C保存，可使用一周。3. 4.3 测定3.4. 3. 1 平板制备将培养基E熔化并冷却至50C士1C，加入适量的菌液，使之充分混合后.取8mL注入灭菌平皿中，保持水平，待其凝固后备用。测定
9、前，须先用0.05，u.g/mL及O.2g/mL的维吉尼霉素标准工作液进行预测试，经培养后，2 SN /T 1134-2002 O. 05g/mL标准工作液所呈现的抑菌圈直径在10mm15 mm，而0.21.lg/mL标准工作液所呈现的抑菌圈直径在15mm20 mm，该菌液浓度为最佳。3. 4. 3. 2 标准曲线的绘制用0.025问/mL、0.05吨/mL、O.1月/mL、O.2问/mL、O.4g/mL和O.8g/mL共六个浓度的维吉尼霉素标准工作液，其中用于最低浓度(0.025问/mL)的三个检定平板作为产生阴性结果的对照，并以O.2g/mL作为参考法度。标准曲线上的每个标准浓度取三个检定
10、用平板为一组。在每个检定用平板上放置六个牛津杯，其中三个加满参考浓度降液，另三个加满标准浓度溶液，参考浓度与标准浓度恪液要间隔放置，五个标准浓度共用15个检定用平板，含量最低的标准浓度的三个检定用平板作为阴性结果的平板对照，其他12个检定用平板绘制标准曲线。将陶瓦盖盖好，4C放置1h2 h后于30C士1C培养17h:l:1h，最后在36C :l: 1 C培养6h，然后翻转平板，除去牛津杯，精确测量产生的抑菌圈直径(精确到O.lmm)，用所有45个参考浓度抑菌圈直径的平均值与各平板参考放度抑菌圈直径平均值的差值作为每个平板的校正值，来校正其他各标准浓度的抑菌圈直径的平均值。将校正后的值，用式(1
11、)、式(2)算出L和H，在半对数坐标纸上以抑菌圈直径(mm)为横坐标(算术级)，以浓度g/mL为纵坐标(对数级)绘制标准曲线。L = 3a + 2b十c- e 一5 . ( 1 ) 日3e+ 2d + c - a -D ,( 2 ) 式中:L一一标准由线上的最低浓度(0.05g/mL)抑菌圈直径，单位为毫米(mm);H一-标准曲线上的最高浓度(0.8g/mL)抑菌圈直径，单位为毫米(mm);C一一标准曲线中全部参考浓度抑菌圈直径的平均值，单位为毫米(mm);a，b、d、一一一分别表示标准曲线中其他浓度标准工作液(0.05吨/mL、O.1吨/mL、O.4g/mL、O. 8月/mL)的抑菌圈直径经
12、校正的平均值，单位为毫米(mm)。3. 4. 3. 3 样液的测定每个样液用三个检定平板，每个平板平均置六只消毒的牛津杯，间隔注满样液和O.2月/mL维吉尼霉素标准工作液，4C放置1h2 h后，于30C士1C培养17h士1h，最后在36C :l: 1 C下培养6h。3. 5 结果的计算和表述培养后，测量抑菌圈直径，如样液抑菌圈直径小于12mm，即报告为阴性;如大于等于12mm，分别求出三个平板中O.2g/mL标准液及样液产生的抑菌圈直径平均值，经校正并查标准曲线，再用式(3)计算试样中维吉尼毒素的含量。c-m 一X ,. ( 3 ) 式中:X 试样中维吉尼霉素的含量，单位为毫克每千克(mg/k
13、g); c 从标准曲线上查出样?夜所对应的抗生素浓度，单位为微克每毫升(f.lg/mL); m 最终试液所代表的样品浓度，单位为克每毫升(g/mL)。注:如测定结果呈阳性，即抑菌圈直径平均值大于等于12mrn时，需进行确证试验(参见附录凹，以证明抑菌物确系维吉尼霉素。3 SN/T 11342002 4 测定低限、回收率4.1 泪tl定低限本方法的测定低限为0.05mg/kg。4.2 回收率鸡肉中维吉尼霉素添加浓度及其回收率的实验数据:0.05 mg/kg时，回收率为80.0%; 0.1 mg/kg时，回收率为81.0 % ; 0.2 mg/kg时，回收率为81.3%。SN/T 1134二200
14、2附录A(规范性附录)培养基成分及制备方法A1 培养基I蛋白陈10.0 g 牛肉膏5.0 g 氧化饷2.5 g 琼脂12.0 g 水1 000 mL 将上述各成分于水中加热溶解，用1mol/L氢氧化铀溶液或10%盐酸调节pH，使其灭菌后pH值为6.5士0.1，分装于试管或克氏瓶内，于121C高压灭菌15min，制成斜面备用。A2 培养基E蛋白陈10.0 g 牛肉膏5.0 g 氯化饷2.5 g 水1000 mL 将上述各成分溶于水中加热溶解，用1mol/L氢氧化铀溶液或10%盐酸调节pH，使其灭菌后pH值为7.0:1:0.1，分装于试管内，121C高压灭菌15mi口。A3 培养基E蛋白陈10.0
15、 g 牛肉膏3.0 g 氧化铀5.0 g 琼脂12.0 g 水1 000 mL 将上述各成分于水中加热溶解，用1mol/L，氢氧化铀溶液或10%盐酸调节pH，使其灭菌后pH值为6.7土0.1，分装于三角瓶内，121C高压灭菌15mi口。5 SN/T 11342002 附录B(资料性附录)确证试验确证试验采用生物自显影法，用标准品作对照，求品值。微量注射器:50L。薄层板:硅肢，Q/YT257一85SG，5cmX20 cm，使用前110C活化2h。展开剂:三氯甲:境十甲醇十乙酸(86十12+2)。展开槽:240cmX57 cmX32 cm。点样量:20L。民方形培养皿:20cmX10 cmX5
16、cm。展开:在饱和槽内进行。检测:取经活化的薄层板，在其下端2cm处划一条基线，然后在该线上分别取20fLL试液和0.20 f-J.g/mL浓度的维吉尼霉素标准溶液，两点相距2.5mm，置展开槽中，用展开剂进行展开，直至溶剂前沿线距板顶端1.5cm处为止。取出薄层板，待风干后放入经高压消毒的长方形培养皿中，将己熔化并冷却至50C :1: 1 C的培养基E均匀地喷在表面，然后用10mL上述己适量接种菌悬液的培养基，铺满整个薄层板，待其凝固，4C放置1h2 h后于30C士1C培养17h士1h，最后在36C:1:1C下培养6ho 经培养后，在薄层上与维吉尼霉素标准液产生抑菌固的Rf值相同位置上，显现
17、抑菌国者即为阳性。6 SN/T 1134-2002 Foreword The method of determination in this standard is adopted in accordance with the Methods for de termination of residues in animal product and sea food edited by Japan Ministry of Hea!th and dra-rted on the basis of experment and verification. Annex A of this standard
18、 is annex of the standard. Annex B of this standard is annex for informa tlon. This standard was proposed and administrated by National Regulatory Commission for Certifica tion and Accreditation. Drafting unit of this standard: Tianjin Exit - Entry Inspection and Quarantine Bureau. The main drafters
19、 o-r this standard: Wang Suqin He Li n Sun Li Chen Zihong. This standard is a prcfessio门alstandard of entry - exit inspection and quarantine promulgated fO I the first !ime啤Note: This English version , a translation from Chinese text , is solely for guidance. 7 SN/T 1134-2002 Method for the determin
20、ation of virginiamycin residues in meats and meat products for import and export -Cylinder method 1 Scope This standard specifies the method of sampling , sample preparation and determination by cylinder method of virginiamycin residues in meats and meat products for import and export. This standard
21、 is applicable to the determination of virginiamycin residues in chicken meat for import and export. 2 Sampling and sample preparation 2.1 Inspection lot The quantity of an inspection lot should not be more than 2 500 packages. The characteristics of the cargo within the same inspection lot, such as
22、 packing , mark , origin , specification and grade should be the same. 2.2 Quantity of sample taken Number of packages Minimum number of in each inspection lot packages to be taken 1-25 26-100 5 101-250 10 251-500 15 501一1000 17 1 001-2500 20 2.3 Sampling procedure A number of packages specified in
23、2.2 are taken at random and opened one by one. From each at least one bag shall be taken as a primary sample. In case no small bag or the content of the bag exceeds 2 kg , cut out a part from the meat in each package of not less than 100 9 with a sharp knife (disinfected with alcohol). Mix the pa叫so
24、f the meat as the mixed primary sample , which shall not be less than 2 kg. Place in a clean container, seal , labeled and send to the laboratory in time. 2.4 Preparation of test sample A representative sample is taken from the combined primary samples , blended , mixed well. Reduce the total amount
25、 of primary to no less than 500 9 as test sample. Place in a clean container which 8 SN/T 1134一2002is then sealed and labeled. 2.5 Storage of test sample The test sample should be frozen and stored at - 18C Note: In the course of sampling and sample preparation , precaution must be taken to avoid co
26、ntamination or any factors wh ich may cause the change of residue contents. 3 Method of Determination 3.1 Principle Virginiamycin residues in sample is extracted with ethanol. After homogenizing and centrifuging , take out suppernatant as test solution for determination by cylinder method , calculat
27、e residues content in sampie through standard curve. 3.2 Reagents and materials Unless otherwise specified , all reagents used should be analytical pure , water is distilled water. Virginiamycin standard and standard solution is caculated by potency. 3.2.1 Ethanol absolute. 3.2.2 Methanol absolute.
28、3.2.3 Methanol+Water:(1 +2). 3.2.4 Physiological saline:Weigh 8.5 9 sodium chloride dissolve in 1 000 mL water, autoclave at 121(; for 15 min. 3圄2.5Potassium dihydrogen phosphate. 3.2.6 Sodium hydrogen phosphate. 3.2.7 Phosphate buffer (pH6. 0) : Weigh 3.5 9 potassium dihydrogen phosphate (accurate
29、to 0.1 g) , dissolve in 400 mL water, weigh 3.0 9 sodium hydrogen phosphate (accurate to 0.1 g) and dissolve in 400 mL water, combine two solution and make to 1 000 mL. 3.2.8 Virginiamycin standard. 3.2.9 Virginiamycin Standard stock solution: Weigh proper quantity of virginiamycin standard , dissol
30、ve in methanol and make to 50 mL, make to 1 000g/mL virginiamycin standard stock solu tion at the end. Store in refrigerator could be used within one week. 3.2.10 Virginiamycin standard working solution: Pipette proper quantity of standard stock solu tion and dilute with phosphate buffer solution (p
31、H6.0) to make to concentration of 0.025g/mL， 9 SN/T 1134-2002 0.05g/mL，0.1g/mL，0.2g/mL，0.4g/mL and 0.8g/mL standard working solution , all the above solution should be prepared daily. 3.2.11 Test strain: Corynebacterium Xeroxes. 3.2.12 Medium 1 (See Annex A1). 3.2.13 Medium II (See Annex A2). 3.2.14
32、 Medium m (See Annex A3). 3.3 Apparatus and equipment 3.3.1 Vernier caliber: measuring range 0 mm 200 mm , precision 0.02 mm or use measurer. 3.3.2 Oxford cup:height 10 mm:!:0.1 mm , inner6.0 mm:!:0.1 mm , extraI8.0 mm:!:0.1 mm. 3.3.3 Centrifuge:no less than 3000 r/min. 3.3.4 Incubator: 30C土1c , 36
33、C :!: 1C , keep horizontal. 3.3.5 Autoclave sterilizor. 3.3.6 Blender:no less than 3000 r/min. 3.3.7 Homogenize: no less than 10 000 r/min , with homogenous cup of 250 mL. 3.3.8 Culture dish: inner 90 mm glass or plastic culture dish with smooth bottom with clay cover. 3.3.9 Other:glass plate，pipe忧e
34、，and capillary pipe. 3.4 Determination procedure 3.4.1 Preparation of sample solution Accurately weigh 10 9 sample (accurate to 0.1 g) in a homogenous cup , add 10 mL ethanol and homogenize for 2 min. Then transfer into centrifugal cup , Centrifuge at 3000 r/min for 20 min , and take out the superna
35、tant as sample solution for determination. 3.4.2 Preparation of strain solution After sterilization of the ampoule of the strain (3.2.11) , cut off the top and add small amount of medium II to dissolve the strain , then transfer into a test tube with medium II , mix well and incu bate at 370C :!: 10
36、C for 24 h. Strike slant of on medium 1 and incubated 37 C :!: 1C for 24 h. 。ul SN/T 1134-2002 Wash down the strain with sterilized physiological saline and transfer into sterilized test tube，由isstrain solution could be used within one week under refrigerated storage. 3.4.3 Determination 3.4.3.1 Pre
37、paration of plate Melt the medium m and cool to 50 C :!: 1C , add proper quantity of strain solution and mix thor oughly, pour 8 mL into each sterilized culture dish , keep horizontal for solidification. Before deter mination , it should be pre - tested with 0.05g/mL and 0.2g/mL standard working sol
38、ution. Af ter incubation , when clear inhibit zones appear with diameter of 10 mm - 15 mm for 0.05g/mL standard solution and 15 mm - 20 mm for 0.2g/mL， this strain solution is suitable for determina tion. 3.4.3.2 Preparation of standard curve Use a series of standard working solution with concentrat
39、ion of 0.025g/mL， 0.05g/mL、0.1g/mLO.2g/mL，0.4g/mL and 0.8g/mL， use three plates for the lowest concentration (0.025g/mL)as negative control and use standard working solution of 0.2g/mL as reference. To prepare standard curve , use tree plates for each standard working solution .In each plate , place
40、 six Oxford - cup with three of them filled with standard working solution of reference concentra tion and others filled with standard working solution of each concentration , place them at inter vals. There will be fifteen plates for all five concentrations , three plates for the lowest concentrati
41、on are used for negative control , and other twelve plates are used for plotting of standard curve. Cover them with caps , store at 4C for 1 h - 2 h , then store at 300C :!: 10C for 17 h:!: 1 h , at the end , store at 360C :!: 1C for 6 h. After incubation invert the plates to remove the cylinders. M
42、easure the diameter of each zone of inhibition as accurately as possible (to the nearest 0.1 mm) . Average the readings of the 0.2g/mL concentration and the readings of the other con centration on each set of three plates. Also average all 45 reading of the 0.2g/mL concentration as the correction po
43、int for the curve. The difference between the total (45 readings) average of 0.2g/mL reference concentration and average of the reference concentration of each set is the correction value for each set. Corrected the average value obtained from other concentration of each set. Use these corrected val
44、ues. Calculate the values of point L and point H according to formula (1) and (2) , and plot the point L and point H on the semilog graph paper. using logarithmic scale for concentration (p.g/mL) as abscissa and arithmetic scale for average zone diameters (mm) as ordi nate. Construct a straight line
45、 through the point L and H as the standard curve. Where 3 a+2b+ c- e E ( 1 ) H = 3e+2d_+ c- a 5 .( 2 ) L-the inhibit zone diameter of the lowest concentration (0.05g/mL) of the standard curve，盯1m;H-the inhibit zone diameter of the highest concentration (0.8g/mL) of the standard 11 SN/T 1134-2002 cur
46、ve，内1凹1; C一theaverage of all inhibit zone diameters of the reference concentration of standard curve，阿1m;a, b , d , e-the corrected average inhibit zone diameter for each of the other concentrations (0.05g/mL，0.1g/mL、0.4g/mLand 0.8g/mL) used for the standard curve , 门1内1.3.4.3.3 Determination of tes
47、t solution Use three plates for each sample and six Oxford cups in each plate , fill the cups with sample solu tion and 0.2g/mL virginiamycin standard working solution at interval , store at 4C for 1 h - 2 h , then store at 30C :t 1C for 17 h :t 1 h , at the end , store at 36C :t 1C for 6 h. 3.5 Cal
48、culation and expression of result 3.5.1 Calculation of result After incubation , measure the diameters of Inhibit zones , If the diameter of inhibition zones caused by the sample solution is 12 mm report as negative . If the diameter of inhibition zone ;: 12 mm , find the mean values of diameters of
49、 inhibit zones caused by 0.2g/mL standard solution and Sample extraction respectively. After calibration , obtain the concentration of sample solution on standard curve ,calculate contents of virginiamycin in test sample according to the formula (3) Where X-contents of virginiamycin , mg/g; X=互m ( 3 ) c-concentration of antibiotic of sample extraction corresponding to the standard ob
