1、EEU 中华人民共和国出入境检验检疫行业标准SNjT 1958-2007 进出口食品中伏马毒素B1残留量检测方法酶联免疫吸附法Screening method of fumonisin B1 residues in foodstuff for import and export-Enzyme Iinked immunosorbent assay method 2007-08-06发布2008-03-01实施二手军队主描刷刷丁中华人民共和国发布吧V国家质量监督检验检疫总局前言本标准附录A、附录B和附录C均为资料性附录。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z中华人民共和国天
2、洋出入境检验检疫局、天洋科技大学。本标准主要起草人2郑文杰、王硕、张宏伟、姚霞、魏亚东、刘炬、张燕、张骏。本标准系首次发布的出入境检验检疫行业标准。SN/T 1958-2007 I 1 范固进出口食品中伏马毒素B，残留量检测方法酶联免疫吸附法本标准规定了食品中伏马毒素B1残留量的酶联免疫吸附测定方法。SN/T 1958-2007 本标准适用于玉米、大米、黄米、燕麦、大麦及花生中伏马毒素B1残留量的筛选检测。2 规范性引用文件下列文件中的条款通过本标t的引用而成为本标准的条款。凡日期的引用文件，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
3、是否可使用这些文件的最新版本凡是不注明一日期的号用文件.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存在雪/ 3 测定方法/ / 3. 1 方法提要/ /J 用甲醇/水提取样品中伏马毒捺B1，)用竞争性酶联免疫吸附法进行检测。3.2 试剂和材料I 1 除另有要求外，所有试剂均为如挺纯品本主臼且工4凶二L规定由如牛级水。3.2. 1 伏马毒素B1酶联免疫吸附测定试剂盒1)(参见3.2.2 甲醇。3.3. 1 3.3.2 天平(精确到0.001g)。3.3.3 旋涡混合器。3.3.4 气浴摇床。3.3.5 酶标仪(测定波长450nm)。3.3.6 洗板机。3.3.7 单道微
4、量加样器，20L，100L，200Lo!)伏马毒素B，酶联免疫吸附测定试剂盒是由天津生物芯片技术有限责任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同的效果，则可使用这些等效产品。SN/T 1958-2007 3.3.8 多通道微量加样器，200L。3.4 试样制备取样品约500g j昆匀，均分成两份，装入洁净容器内作为试样，密封，并标明标记。3.5 试样保存将试样子4C冷藏保存。在制样的操作过程中，应防止样品受到污染或发生残留物含量的变化。3.6 提取将玉米等谷物样品各取25g充分粉碎，从中取样品各5g，加入20mL甲醇/水(
5、3 1，体积比)，振荡萃取15min，静置5min，取部分上清液用掩蔽弗l稀释15倍后进行ELISA检测。3. 7 酶联免疫法检测3.7. 1 标准曲线的制备用溶液I稀释10mg/L的伏马毒素B:标准溶液储备液至100g/L，用溶液I对100问/L的伏马毒素B:标准溶液进行1 3梯度稀释，得到一系列不同浓度的伏马毒素B:标准溶液(100f1 g/L, 33.3g/L，11.1g/L，3.7g/L，1. 2g/L，0.4g/U。3.7.2 样品测定在酶标板中分别加人100L不同浓度的伏马毒素B:标准溶液和样品待四液。在空臼和对照孔中分别加入100L的水。每个标准溶液、样品待测液、空白和对照孔均做
6、平行。空白孔中加入100L磷酸盐缓冲液。除空白孔外，在每个孔中则加入100L的酶标记物稀释液。轻拍混匀，用粘胶纸封住微孔以防溶液挥发，室温孵育60mino弃掉酶标板中的液体，用洗涤液洗板三次。加入混合好的底物液150L，轻轻混匀，室温放置30min后，每个孔加入50L的终止液，轻轻晃动酶标板，10min内在酶标仪上读出每孔450nm吸光值。3.8 空白试验除不加试佯外，均按上述测定步骤进行。3.9 结果的计算3.9. 1 计算抑制率值不同浓度伏马毒素B:对抗原抗体结合反应的抑制率计算见式(1), ,. Aw.c. -AR白寸IC二11ff-u.主且Ix100% L. A对照A空白. ( 1 )
7、 式中zIC-伏马毒素B:对抗原抗体结合反应的抑制率，灿A对用不加入伏马毒素B:标准液，仅加入酶标记稀释物和磷酸盐缓冲液，在450nm下测得的平均吸光度值pA tl:.Pu 伏马毒素B:标准液或样液在450nm下的平均吸光度值;A空白一一不加入酶标记稀释物及伏马毒素B:标准液，仅加入水和磷酸盐缓冲液，在450nrn下测得的平均吸光度值。3.9.2 绘制标准曲线以抑制率为纵坐标，伏马毒素B:浓度为横坐标(横坐标取对数刻度)绘制标准曲线。每次试验均应重新绘制标准曲线，参见附录C。3.9.3 结果计算从标准曲线上读取样液抑制率所对应的伏马毒素B:浓度(c)，按式(2)计算试样中的伏马毒素B:残留量:
8、X cXR ( 2 ) 式中X 试样中伏马毒素B:残留量，单位为微克每千克(g/kg); 2 SN/T 1958-2007 c一一一根据佯品孔的抑制j率查得试样中伏马毒素B，浓度，单位为微克每升(g/Ll; R一一样品换算系数，其数值为60。4 检测低限、回收率4. 1 检测低限本方法检测低限为12g/峙。4.2 回收率伏马毒素B，添加浓度和回收率的实验数据参见附录B。实验结果为阳性应用仪器方法进行确证。3 SNjT 1958-2007 附录A(资料性附录伏马毒素，酶联免在测定试剂盒本标准目酶5联免疫测定步骤中使用的试剂盒为天津生物芯片技术有限责任公司产品a) 9仍6孔自酶每标板gb) 伙马毒
9、素B，酶标记物溶液;c) 底物液A:于4C保存，使用时需达到室温;d) 底物液B:使用前室温避光保存.使用时，按底物液Az底物液且为32: 1 (体积比)混合使用;e) 反应终止液$f) 掩蔽剂2于4C保存;g) 磷酸盐缓冲液2于4C肮7使用时丁洛阳i!i3IJ拙，用仄1: 5稀释;h) 洗涤液z使用时，用水1: 5稀释o/ 严/ JJ/ 飞飞tj/ TFv YZT14143zyw? 4441vpv HiJF Y 2)给出该信息是为了方便本标准的使用者.并不表示对某一产品的认可。如果其他产品具有相同的效果.需经实阶评估后使用这些等效产品.4 S:-;jT 1958一2007附录B(资料性附录)
10、伏马毒素8，添加浓度和平均回收率表8.1伏马霉素8，添加浓度和平均回收率样品添加浓度/(问/kg)平均回收率/%12 100 王来250 104 500 86 12 100 一-一一一大米250 114 110 100 黄米82 93 h 12 100 大麦250 102 一一一一u 阻，他t皿_.山四邮叶。0-，-甲叩阳阿甲耳104 100 燕麦88 87 100 花生f i dZ5 94 L j 飞00J / I I 91 1 5 SN/T 1958-2007 附录(资料性附录)伏马霉素，标准曲线C 100 90 80 70 60 50 40 30 20 10 汉得军军10 0.1 FB，
11、放度/(ng!mL)。0.01 伏马毒素，标准曲线图C.16 SN/T 1958-2007 Foreword Annex A , B and C of this standard are all informative annexes. This standard was proposed by and is under the charge of Certification and Accreditation Administra tion of the Peoples Republic of China This standard was drafted by Tianjin Entry-
12、Exit Inspection and Ouarantine Bureau of the People s Re public of China. , Tianjin University of Science and Technologuy. This standard was mainly drafted by Wenjie Zhe吨，Shuo Wa吨，Hongwei Zhang , Xia Yao , Yadong Wei , Xuan Li u, Yan Zhang , Jun Zhang. This standard is a professional standard for en
13、try-exit inspection and quarantine promulgated for the first time. 7 SN/T 1958-2007 Screening method of fumonisin 81 residues in foodstuff for import and export -Enzyme linked immunosorbent assay method 1 Scope This standard specifies the meth foodstuff etermrr奇Tb百h飞囚7口l1etj-，odof fumonisin B, resid
14、ues in -.:.=.一This standard is applicable to the screen of furr飞on门nB, residues)n corn. barley. rice. oat. peanut and sorghum. PO日tiveresults should be confi; by anoth5rethod. F / , ? ./ 2 Normative references /: . The foliowing standard contain ptovisions which. through referencq in this text. cons
15、titute provi . sions of this standard. Parties to与greementsbased vn吁his，tandarl审萨3 Method of inspection he tefis the competitive enzyme-linked 川二isof 3. 1 Principle of determination immunoreaction. 3.2 Reagents and materials In this standard. all the chemical reagents should be analytically pure and
16、 water is the first-degree water as GB/T 6682 described. 3.2.1 Fumonisin B, detection kit (see Annex Al. 1) Fumonisin 81 detection kit is the commercial name which was provided bthe Tianjin Biochip Technotogies Co. , Ltd This information beng given is convenient for the user of the standard and is n
17、ot the confjrm of 50me products pro toco l. Other products protocol should be confirmed bexperiments if any difference. 8 3.2.2 Methanol. 3.2.3 Na,HPO, 12H,O. 3.2.4 NaH,PO, 2H,O 3.2.5 NaCI. 3.2.6 0.5% FG-PBS dilute. 3.2.7 3.2.8 Solution 1. SN/T 1958-2007 旷ml/ .;/ AII solution must be stored at low t
18、可节leratureand Jver be placed at room temperature longer than 4 hours: reach room tem口erature却缸。reuse. /. f. - .，帽-睛-噜-嗣蝇回3.3 Apparatus and equipment I 3.3. 1 Muller. 3.3.2 Balance 3.3.3 Vortex mixer 3.3.4 Vibrator. 3.3.5 Microwell system. J / 3.3.6 3.3.7 Pipettes, 20L. 100L.200L 3.3.8 Multichannel p
19、ipette, 200L. 3.4 Preparation of test sample The combined primary sample is reduced to 500 g. which is blended. homogenized thoroughly. and then divided into two equal portions. Each portion is placed in a clean container as the test sample. which is sealed and labeled 9 SN/T 1958-2007 3. 5 Storage
20、01 test sample The test sample should be stored at 4 t. In the course 01 sampling and sample preparation , precau tion must be taken to avoid the contamination or any lactors which may cause the change 01 residue content. 3. 6 Sample extraction Samples were linely ground with a laboratory blender an
21、d add 20 mL methanol/H,OC3 1, v/v) to 5 9 dried sample , shaking extraction lor 15 min , standing lor 5 min , upper clear solution samples were diluted 15 lold with 0.5% FG-PBS. 3.7 ELl SA determination 3.7.1 Manulacture 01 calibration curve Dilute the prepared standard solution with solution 1 into
22、 100g/L， 33.3g/L. 11.1g/L. 3.7g/L， 1.2g/L，0.4g/L 3.7.2 Detection 01 sample Insert a sufficient amount 01 microtitre wells into the microwell holder lor all standards and samples to be run in duplicate. Record standard and sample positions. add 100L 01 six kinds 01 prepared lu momsln 队standardsolutio
23、n and sample solution to separate duplicate wells. Add 100L double wa ter as blank. Add 100L 01 diluted lumonisin B, enzyme conjugate Cused within 8 h) to the bottom 01 each well and add 100L PBS in blank. Ilap lightly and mix thoroughly. Sealing all wells with adhesive paper to avoid solution evapo
24、rating , incubate lor 60 min at room temperature. Pour the liquid out 01 the wells and wash the wells three times with PBST. Add 150L 01 substrate and chromogen to each well CTake care the substrate solution in the dark) Ila口lightlyand mix thoroughly and incubate lor 30 min at room temperature. Add
25、50L 01 st。口solutionto each well , Ilap lightly and mix thoroughly. Measure and record the absorbance value 01 each well solution at 450 nm within 10 min. 3. 8 Blank test The operation 01 the blank test is the same as that described in the method 01 determination but without addition 01 sample 3.9 Ca
26、lculating the result and express 3.9. 1 Calculating inhibition value in percentages Ca lculate the inhibition value 01 the combination口1each lumonisin B, standard and antigen-antibody, the lumonisin B, standard and sample inhibition value are calculated in percentages according to the 10 SN/T 1958-2
27、007 formula( 1) : ,_ A一- Ah , l IC = I 1 一旦旦旦IX 100% L. Antrol一A刷刷) 1 ( . . . . . . . . . . . . . . . . . . . . . . . . where IC-the inhibition value of fumonisin 8, to the response of antigen and antibody, %: A脚时0g/L fumonisin 8, standard mean absorbance value in percentages; A，盯p，.-fumonisin8, sta
28、ndard or sample solution mean absorbance value , Ab1a附0g/Lfumonisin 8, standard solution and enzyme conjugate mean absorbance value. 3.9.2 Plot calibration curve Make the inhibition value in percentages(arithmetic grade)as ordinate,and fumonisin 8, concentra. tion as abscissa , plot the calibration
29、curve of the inhibition value in percentages and fumonisin 8, standard concentration(see Annex C). A new calibration curve should be prepared for each determi nation. 3.9.3 Calculating the result The fumonisin 8, concentration of the test sample solutin can be read corresponding to the percent age i
30、nhibition value of each sample from calibration curve and namely equal to the residue content of fumonisin队inthe test sample(g/L). X=cXR . ( 2 ) where X -the residue of fumonisin 8, (g/kg) , c-the residue of fumonisin 8, read from inhibition value(g/L) , R一thesample convertion factor:60 4 Limit of d
31、etermination recovery and precision 4. 1 Li mit of determination The limit of determination of this method is 12g/kg. 4. 2 Recovery The experimental data of these fortifying concentrations of fumonisin 8, and average recoveries are as Annex 8. The positive result should be confirmed by the equipment
32、 method. 11 SNjT 1958-2007 Annex A Clnformative Annex) Fumonisin 8, detection kit Fumonisin B, detection kit is provided by the Tianjin Biochip Technologies Co. ,Ltd2). It includes, a) 96-microwell plate; b) Fumonisin B, enzyme conjugate; c) Substrate solution A; d) Substrate solution B, Store t dar
33、k place. Substrate A Sub-strate B 32 1; e) Stop solution; f) 0.5% FG-PBS.Store at 4C; g) Phosphate Buffered Saline( P h) 2) The information supplying herein is just for the convenience usage for the users of this standard and not for 50me certain products authorization. The testin9 process of anothe
34、r equivalent kits maybe differ,so it should be used af tertesting , evaluation andverification 12 Sample corn rlce sorghum barley 。atpeanut Annex B (Informative Annex) Addition and average recovery of fumonisin B, Table 8. 1-Addition and average recovery of fumonisin 8, SN/T 1958-2007 Addit旧们!(g/kg)
35、Average recoverv/% 12 100 250 104 500 86 12 100 250 114 45，0口、Y 110 /2 、/矿100 / 250 / 82 1/ 5/ 080 93 f2 1口D250 102 5口。104 j .，、100 / 88 J 87 I 12 i 1口。/ f 250 ! 94 500 J 91 t J、Jf 13 SN/T 1958-2007 Annex C (Informative Annex) Standard curve of fumonisin 8, 100 90 80 70 60 50 40 30 20 10 waz。ZRA-ZZ【
36、10 0.1 Concentration of FB1 !(ng/mU Figure C. l-Standard curve of fumonisin B, 。0.01 14 li SN/T 1958-2001 中华人民共和国出人搅检验检疫行业标准迸出口食品中伏马毒素肌残留量检测方法酶联免疫吸附法SN/T 1958-2007 幡中国标准出版社出版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 开本880X12301/16 印张1.25 字数25千字2007年11月第一版2007年11月第一次印刷印数1-2000睡书号，155066 2-18240 定价12元
