1、中华人民共和国出入境检验检疫行业标准SN/T 1573-2005 贝类中神经性贝类毒素检验方法小鼠生物法Inspection of neurotoxin shellfish poison in shellfish一Method of mouse biology 2005-05-20发布2005-12-01实施中华人民共和国发布国家质量监督检验检疫总局前言本标准的附录A为规范性附录。本标准由国家认证认可监督管理委员会提出并归口。本标准主要起草单位:中华人民共和国辽宁出入境检验检疫局。本标准主要起草人:赵昕、曹际娟、秦成、宋慧君、于坷、唐守亭。本标准系首次发布的出入境检验检疫行业标准。SN/T 1
2、573-2005 I 1 范围贝类中神经性贝类毒素检验方法小鼠生物法SN/T 1573-2005 本标准规定了贝类及其制品中神经性贝类毒素检验的抽样、制样和小鼠生物检测方法。本标准适用于海产双壳类贝肉、贝柱和其他可供食用部分的神经性贝类毒素的检验。2 术语和定义下列术语和定义适用于本标准。2. 1 神经性贝类毒素neurotoxin shellfish poinson, NSP 化学结构以短裸甲藻毒素(brevetoxin-2)为代表的，摄食后可产生神经性中毒和消化道症状的存在于贝肉内的海洋生物毒性物质的总称。2.2 小鼠单位mouse unit,MU 对体重为20g的小白鼠腹腔注射1mL贝类
3、提取液后，在5min时致死白鼠所需的最低毒素量。2.3 申位数median data 把变量值按大小次序排列，居于中间位置的那个数值就是中位数。3 抽样和制样3. 1 检验批以不超过100t为一检验批，同一检验批的商品应具有相同的特征，如产地、季节、规格、包装和标记等。3.2 抽样敢量根据各检验批的重量，按表1确定抽样点(件)数。如为散装的应均匀抽样。表1样晶抽样点(件)数的确定检验批重量/t抽样点(件)数混合样品数10以下10 2 1l50 15 3 51100 20 4 3.3 抽样方法按4.2规定的抽样点(件)数，随机抽取样品。从每5个抽样点(件)抽取的原始样品组成一个泪合样(依受检贝类
4、品种决定样品的采集量，应保证其可检部分重量不少于1000 g) ，装入清洁容器内，加封标记后，及时送交实验室检验。3.4 样晶制备3.4. 1 实验室样晶制备分析样品要有充分的代表性，即从1昆合样品中挑选良好的贝类个体去壳取肉，去壳肉量应达到SN/T 1573一20051 000 g，取其中200g作为检样，另800g作为复检样、备检样和留样。新鲜贝类如果不能及时检验，按3.4.2.1方法将贝肉分离，将100mL盐酸(0.1 mol/L)中加入沥水后的200g贝肉，置于40C冷藏保存，备检。3.4.2 试样制备3.4.2.1 牡蜻、蛤及贻贝用清水将贝壳外表彻底洗净，切断闭壳肌，开壳，用清水淋洗
5、内部去除泥沙及其他外来物。将连接在交合部的组织分离，仔细取出贝肉，避免割破肉体。开壳前不得加热或用麻醉剂处理。收集200g肉置于10号筛子中沥水5min(避免贝肉堆积)，检出碎壳等杂物，将贝肉均质。3.4.2.2扇贝取贝肉用作检测。沥干及均质过程同3.4.2.1。3.4.2.3 贝类罐头将罐头内所有内容物(肉及液体)倒人均质器充分均质。如果是500g以上大罐，将贝肉沥水并收集沥下的液体，分别称量，将固形物和汤汁按原比例混合，充分均质。3.4.2.4 用酸保存的贝肉沥去酸液，将沥干的贝肉充分均质。3.4.2.5 冷冻贝类在室温下，使冷冻的样品(带壳或脱壳的)呈半冷冻状态，按3.4.2.1方法开壳
6、、淋洗、取肉、均质。3.4.2.6 贝肉干制晶干制品可于等体积盐酸(0.1mol/U溶液中40C下浸泡24h48 h，按3.4.2.4方法沥干、均质。3.5 试样保存上述3.4中均质处理的样品如不能及时检测，可取100mL盐酸(0.1mol/U搭液100g加入巳均质贝肉中，置于40C冷藏保存(尽可能及时检验)。4 测定方法4. 1 方法提要本方法采用小鼠单位定量测定NSP毒素的总量，采用brevetoxin-2作为毒素的标准品。根据小鼠注射贝类提取液后的死亡时间，查出鼠单位，并按小鼠体重，计算确定每克贝肉内NSP的小鼠单位。所测定结果代表存在于贝肉内各种化学结构的NSP。4.2 试剂和材料4.
7、2.1 丙酣(分析纯)。4.2.2 二氯甲皖(分析纯)。4.2.3 1%吐温-60生理盐水。4.2.4 无水硫酸铀。4.2.5 小白鼠:体重为20g士1g ICR品系，雄性。4.2.6 标准品:短裸甲藻毒素(brevetoxin-2)。4. 3 设备和器皿4.3.1 旋转蒸发器。4.3.2 真空泵。4.3.3 均质器。4.3.4 化学通风柜。4.3.5 天平:感量为O.1 gO 4.3.6 冰箱:OOC50C和150C-200C。4.3.7 500 mL梨形瓶或圆底烧瓶。4.3.8 烧杯500mL和200mL。4.3.9 布氏漏斗。4.3.10 500 mL抽滤瓶。4. 3. 11 500 m
8、L分液漏斗。4.3. 12 玻璃珠。4.3.13 注射器:2mL或5mL无菌注射器。4.4 测定步骤4.4.1 提取SN/T 1573-2005 取样品100g用300mL丙酣均质1min。用布氏漏斗减压抽滤并收集提取液。残留物再用样品等体积(100mL)丙酣冲洗抽滤两次，合并丙酣抽滤液。将抽滤液转移至旋转蒸发瓶中，560C旋转蒸发除去丙酣。将剩余水相提取物用二氯甲皖冲洗转移到500mL的分液漏斗中，轻轻摇动，静置分层。二氯甲皖层(下层)经无水硫酸铀过滤，滤完用二氯甲烧继续漂洗至无水硫酸铀无色。收集洗脱液移入300 mL旋转蒸发瓶中，400C旋转蒸发后的存留部分即为样品脂质提取物。用1%吐温6
9、0生理盐水将样品脂质提取物定容至10mL，添加玻璃珠振荡使其悬浮棍匀，所形成的悬液即为提取液。4.4.2 小鼠试验将小鼠称量并记录质量。每个样品注射3只小鼠。对每只试验小鼠腹腔注射1mL提取液，同时另取一只小鼠注射1mL 1%吐温-60生理盐水作为试剂空白对照。注射时若有1滴以上提取液溢出，就须将该只小鼠丢弃，并重新注射l只小鼠。记录在射完毕时间，仔细观察小鼠停止呼吸时所表明的死亡时间(到小鼠呼出最后一口气止)，记录死亡时间。若小鼠的中位数死亡时间小于8min，则要用1%吐温-60生理盐水对提取液进行稀释，再注射另一组3只小鼠，以得到大于8min的死亡时间。5 结果计算与报告5.1 NSP毒力
10、的计算与判断根据小鼠的死亡时间，按附录A中表A.1查出相应的每毫升注射液的小鼠单位数，质量校正系数乘以该只小鼠的小鼠单位数再乘以定容体积，即按式(1)计算，求得每克贝肉中小鼠单位数。小鼠单位(MU)/g= 10M X W /100 ( 1 ) 如果小鼠的死亡时间小于8min.就用1%吐温-60生理盐水将提取液稀释，使小鼠的死亡时间达到表A.1中的死亡时间。然后按式(2)计算，求得每克贝肉中NSP毒素的含量。小鼠单位(MU)/g= 10MX W X D/100 ( 2 ) 式中:M二十每毫升注射液的鼠单位数，(MU/mL);W一一重量校正系数;D一一稀释倍数。5.2 结果报告5.2.1 若小鼠的
11、死亡时间大于930min，结果报告为小于0.1MU/g。5.2.2 若小鼠的死亡时间小于930min，结果按照5.1计算得到实际结果报告。3 SN/T 1573-2005 附录A(规范性附录)换算关系表A.1小鼠死亡时间与小鼠单位换算关系死亡时间/min(20g小鼠)小鼠单位/(MU/mL)8 10.0 10 9.0 12 8.0 14 7.0 16 6.0 18 5.0 20 4. 5 30 4.0 38 3.8 45 3.6 60 3.4 83 3.2 105 3.0 140 2.8 180 2.6 234 2.4 300 2.2 360 2.0 435 1. 8 540 1. 6 645
12、 1. 4 780 1. 2 930 1. 0 表A.2小鼠重量与重量校正系数的换算关系小鼠重量/g重量校正系数15 O. 69 16 O. 75 17 0.81 18 0.87 19 O. 94 4 SN/T 1573-2005 表A.2(续)小鼠重量/g重量校正系数20 1. 00 21 1. 06 22 12 23 18 24 1. 24 25 1. 30 26 1. 36 5 SN/T 1573-2005 Foreword Annex A of this standard is an informative annex. This standard was proposed by an
13、d is under the charge of National Regulatory Commission for certi fication and Accreditalion. This standard was drafted by liaoning Entry-Exit Inspection and Ouarantine Bureau of the People s Republic of China. The main drafters of this standard are Zhao Xin ,Cao Jijuan ,Oin Cheng ,Song Huijun , Yu
14、Ke and Tang Shouting. This standard is a professional standard promulgated for the first time. 6 SN/T 1573-2005 Inspection of neurotoxin shellfish poison in shellfish一Method of mouse biology 1 Scope of Application This Standard specifies the methods of sampling , sample preparation and method of mou
15、se biology for inspection of neurotoxin shellfish poison in shellfish and shellfish products. This Standard is applicable to the inspection of neurotoxin shellfish poison in bivalve meat, shellfish maat ,and other edible parts of marine shellfish. 2 Terms and Definitions This Standard adopts the fol
16、lowing terms and definitions. 2.1 Neurotoxin Shellfish Poison(NSP) NSP means all marine biological toxic substances in shellfish meat. which features brevetoxin-2 for chemical constitution and can result in nervous poison and symptom ot digestive tract after being in gested. 2.2 Mouse Unit (MU) MU i
17、s defined as the minimum amount of poison required to kill a mouse weighing 20 9 in 15 min when 1. 0 mL of shellfish extract is injected into abdominal cavity of that mouse. 2.3 Median Data Median Data means the value in the median position when the variable values are arranged in order of slze. 3 S
18、ampling and Sample Preparation 3. 1 Inspection Lot The quality of an inspection lot should not be more than 100 t. The goods in the same inspection lot shall be of the same characteristics such as origin ,season ,specifications,packing and marks. 7 SN(T 1573-2005 3. 2 5ampling Quantit According to t
19、he weight of inspection lot, determine sampling quantity as per Table 1. Implement e ven sampling if the goods are in bulk. Table 1 Determination of 5ampling Quantit Weight of Inspection Lot/t Sampling Point Quantity Quantity of Mixed Samples Below 10 10 2 11.50 15 3 51.100 20 4 3. 3 5ampling Method
20、 Implement random sampling according to the sampling point quantity specified in 4. 2. Combine the original samples from every 5 sampling points to form a mixed sample (determine the quantity of collected samples according to the type of shellfish to be inspected; ensure that the weight of inspec te
21、d parts is not less than 1 000 g) ,and put this mixed sample into c1ean container with proper mark and timeldeliver it to lab for inspection. 3. 4 5ample Preparation 3.4.1 Preparation of Test 5ample The analytic sample should be typical , i. e. select excellent shellfish from mixed samples and remov
22、e the shell to get the meat; the meat without shell should reach 1 000 g , among which 200 9 is to be in spected while the remaining 800 9 is taken as sample to be reinspected or file sample. If the fresh shellfish isn t inspected timely, separate the meat from the shell by use of the method mention
23、ed in 3.4.2.1 ,and add shellfish meat of 200 9 to HCI (0. 1 mol/L) of 100 mL, then store the meat in cold conditions (40C) for inspection. 3. 4. 2 Preparation of 5ample 3. 4. 2. 1 Oyster, Clam and Mussel Thoroughly wash outside of shellfish with clean water, cut adductor to open the shell , then rin
24、se in side with clean water to remove the sand and other foreign materials. 5eparate the meat from shell carefully to avoid cutting or damaging body of mollusk. It is forbidden to heat the shellfish or treat t with anesthetic before opening the shel l. Collect approxmatel200 9 of meat and put t in N
25、o. 10 sieve,and let it drain for 5 min (avoid pilng up of shellfish meat). Pick out and dscard shell pieces; homogenize the shellfish meat. 8 SN/T 1573-2005 3. 4. 2. 2 Scallop Acquire scallop meat for inspection , the drainage and homogenization process is the same as that in 3.4.2.1. 3. 4. 2. 3 Can
26、ned Shellfish Transfer the entire contents (meat and liquid) of the can into homogenizer for thorough homogeniza tion. For large can of more than 500 g , drain the meat and collect all liquid , then determine the weights of meat and volume of liquid. Remix the solid substance and liquid according to
27、 the original proportion and fullhomogenize the mixture. 3. 4. 2. 4 Acid-preserved Shellfish Meat Drain the acid solution from shellfish meat and fully homogenize the drained meat. 3. 4. 2. 5 Frozen Shellfish Meat At the room temperature, make the frozen samples (with or without shelD semi-frozen ,
28、then open the she川，rinse，obtainmeat and homogenize meat according to the method specified in 3.4. 2. 1. 3. 4. 2. 6 Dried Shellfish Meat Products Soak the dried products in HCI (0. 1 mol/L) solution of the same volume for 24 h-48 h at the temper ature of 4C ,then drain and homogenize the products by
29、use of the method in 3.4.2.4. 3. 5 Storage of T est Sample If the homogenized samples in 3.4 are not inspected timely,add 100 9 HCI (100 mL,O. 1 mol/L) solu tion to homogenized shellfish meat , then store the meat in cold conditions C40C) and inspect it as soon as possible. 4 Method of Determination
30、 4. 1 Method Introduction This method adopts MU to determine gross amount of NSP and adopts brevetoxin-2 as the standard poison. Find out the MU according to the death time of the mouse injected by shellfish extract, and determine MU of NSP in shellfish meat of 1 9 according to the mouse weight. The
31、 test results display NSP of various chemical structures in shellfish meat. 4. 2 Reagents and Materials 4.2. 1 Acetone Canalytical pure). 9 SN/T 1573-2005 4.2.2 Dichloromethane Canaltical pure). 4.2.3 1 % tween 60 normal saline. . 2. 4 Anhydrous sodium sulfate. 4.2.5 Mouse:weighing 20 9士1g, ICR stra
32、in , male. 4.2.6 Standard: brevetoxin-2. 4.3 Equipment and Vessels 4.3. 1 Rotary evaporator. 4. 3. 2 Vacuum pump. 4.3.3 Homogenizer. 4. 3. 4 Chemical venti lated case. 4.3.5 Balance:sensibility reciprocal is O. 1 g. 4.3.6 Refrigerator:OoC _50C and一15t-一200C.4.3. 7 500 mL pear-shaped flask or round b
33、ottom flask. 4.3.8 Beaker of 500 mL and beaker of 200 mL. 4.3.9 Buchner funnel. 4.3.10 Suction flask of 500 mL. 4. 3. 11 Separating funnel of 500 mL. 4.3.12 Glass bead. 4.3.13 Injector:2 mL or 5 mL aseptic injector. 4.4 Determination Steps 4. 4. 1 Extract Fetch sample of 100 9 and homogenize it with
34、 acetone of 300 mL for 1 min , implement pressure re-10 SN/T 1573一2005duction and suction with Buchner funnel and collect extracting solution , then rinse and suck the resid uals with acetone (100 mL, the same as volume of sample) for twice and combine the suction solu tion of acetone. Transfer the
35、suction solution to rota叩evaporator，rotate the evaporator in an ang le of 560C to evaporate the acetone. Rinse the remaining water phase extract with dichloromethane and transfer it to separating funnel of 500 mL, slightly shake the funnel to make the solution laminate up on still placement. Filter
36、the layer of dichloromethane (Iower layer) through anhydrous sodium sul fate, then continuously rinse with dichloromethane until anhydrous sodium sulfate becomes color less. Collect the elutriant and transfer it into rotary evaporator of 300 mL, the residuals after rotation (400C) and evaporation ar
37、e the sample lipid extract. Fix the volume of sample lipid extract to 10 mL with 1 % tween 60 normal saline,add glass beads and shake the solution to make beads float evenly, then the formed suspending solution is the extracting solution. 4. 4. 2 Mouse Experiment Weigh the mice and make records. Res
38、pectively inject every sample into 3 mice, inject 1 mL extrac ting solution into abdominal cavity of each mouse, and inject 1 mL 1 % tween 60 normal saline into another mouse for the purpose of non-reagent comparison. If more than one drop of extracting solu tion is spilled out upon injection , disc
39、ard this mouse and reinject extracting solution into another mouse. Record the time when the injection is finished , and carefully observe and record the death time in which the mouse stops breathing (i. e. draws its last breath). If the median datum of mouse death time is less than 8 min , dilute t
40、he extracting solution with 1 % tween 60 normal saline , then in ject such soluton into 3 mice of another group to obtain the death time of more than 8 min. 5 Result Computation and Report 5. 1 Computation and Determination of NSP According to the mouse death time , find out corresponding quantity o
41、f MU in 1 mL injection as per Table A. 1 of Appendix A , multiply weight correction factor by mouse units and fixed volume, i. e. compute according to Formula (1) to determine the quantity of MU in shellfish meat of 1 g. MU/g = 10M x W /100 ( 1 ) If the mouse death time is less than 8 min , dilute t
42、he extracting solution with 1 % tween 60 normal saline to make mouse death time reach death time in Table A. 1, then compute according to Formula (2) to determine the NSP content in shellfish meat of 1 g. Where: M 一一一MU/mL;W一一一Weightcorrection factor; D一一-DilutionRation. MU/g= 10M x川Ix 0/100 ( 2 ) 1
43、1 SN/T 1573-2005 5. 2 Result Report 5.2. 1 If the mouse death time is 930 min. result turns out to be 0. 1 MU/g. 5.2.2 If the mouse death time is 930 pute according to 5. 1 to obtain actual result. 12 SN/T 1573-2005 Appendix A (Normative Appendix) Conversion relationship Table A. 1 Conversion relati
44、onship between mouse death time and MU Death time/min (20 9 mouse) MU/(MU/mL) 8 10.0 10 9.0 12 8.0 14 7.0 16 6.0 18 5.0 20 4.5 30 4.0 38 3.8 45 3.6 60 3.4 83 3.2 105 3.0 140 2.8 180 2.6 234 2.4 300 2.2 360 2.0 435 1.8 540 1.6 645 1.4 780 1.2 930 1.0 Table A.2 Conversion relationship between mouse we
45、ight and weight correction factor Mouse weight/g Weight correction factor 15 0.69 16 0.75 17 0.81 18 0.87 13 SN/T 1573-2005 Table A. 2 (Continued) Mouse weight/g Weight correction factor 19 0.94 20 1.00 21 1.06 22 12 23 18 24 1.24 25 1.30 26 1.36 14 山OON|的hmFH军中华人民共和国出入境检验检疫行业标准贝类中神经性贝类毒素检验方法小鼠生物法SN/T 1573-2005 峙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峰印张1.25 字数25千字2005年8月第一次印刷开本88X 123 1/16 2005年8月第一版晤定价12.00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. 2-16320
