【商检行业标准(SN)】 贝类中神经性贝类毒素检验方法 小鼠生物法(附英文版)

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-Method of mousebiology
2005-05-20发布
华人民共和国
国家质量监督检验检疫总局
2005-12-01实施
本标准的附录A为规范性断录，
本标准由区家认证认可监督管理委员会提出并口，本标滩主要起草单位：中华人民共和国辽宁山人境检验检渡局本标主起草人:赵昕,曹际娟、秦戚、宋薏若、于唐守亭。
本标准系首次发布的出入境检验检疫行业标准。SN/T1573—2005
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具制品户神经性贝类素检验的抽样，制样和小鼠生物检测方法。本标准适压一海产双壳类质肉、贝栏利其他可供食用部分的神经性灭类毒素哟检验。2术语和定义
下列术语利定义适用=本标准，
neuroloxin shellrish poinson,Nsi神经性贝类毒素
化学结构以短裸中藻毒素（breve:oxin-2)为代表的，摄食后i可产生神经性中毒和消化道症状的存在丁贝肉内的海洋生物毒性物质的总称。2.2
mouse unit,MU
小鼠单位
对体重为20只的小白鼠履腔注射」ml.贝类提取液后,在15 mn时致处白鼠所需的最低声素量：2.3
中位数nredian data
把变量值接人小次序排列，店中问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100 t为一检验批,同一检验批的商品应只有相同的特征,如产地,季节,规格,包装和标记等。
3.2抽样数量
慢据各检验批的虽兰·接表1确定抽样（件)数：如为散装的虚每抽样：表1样品抽样点(件)数的确定
检验批重直
1C以下
3.3抽样方法
抑详点(件)数
靓合详品数
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)抽取的原始样品组战一个混合样（依受检贝类品种快定椎品的采集量·应保证其口检部分重量不少于1000g),装人清洁容器内，加封标记所，及村送交实验空检验。3.4样品制备
3.4.1实验室样品制备
分析样品要有充分的代表性，即从混合样品中挑选良好的贝类个本去亮取肉，去壳肉量成达到SN/T 1573—2005
10008,取馬中200g作为检样，另800名作为复检栏、各检样和留样。新鲜典类如果不能及压检验，按3.4.2.1方法将炎肉分离，将10ml.盐酸(C.11ra1/1)中加人沥水后的20℃贝为，置于4℃冷溅保存，备。3.4.2试样制备
3. 4. 2. 1牡蛎、蛤及贻贝
用清水将壳外表御底洗净，切断闭壳肌，开壳用清水淋洗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，避免破肉。开壳前不得加热或用麻醉剂处提，收集200肉置于1C 号筛了中溉水5 1min（避免以肉堆积）、捡出碎壳等杂物.将坝内均质：3. 4.2,2扇贝
取贝用作检测。沥下及均质过程向3.4.2.1，3.4.2.3贝类罐头
将罐头内所有内容物（肉及凌体)倒人均质器充分均质。如果是50以1大罐，将贝肉沥水并收集溯下的腋体.分别称量，固形物和汤汁按原比例湿合，充分均质，3. 4.2.4用酸保存的贝肉
泌去酸液，将泌十的贝肉充分均质，3.4.2.5冷冻贝类
在空温下，使冷瓷的样品（带亮或脱壳的)呈半冷冻状态，按3.4.2.1方法壳、淋洗，取肉，均质3.4.2.6贝肉干制品
干制品可于等体积盐酸(0.1mol/L)溶液中2℃下没池24h～48h，接3.2.2.4方法溯干,均质3.5试样保存
上述3.4户均质处埋的样品如不能及时检浏，可取:0m1.盐酸(0.1:n1/1.)溶液10期人亡均质贝肉巾，置于2℃冷蔽保存（尽可能及时检验）。4测定方法
4.1方法提要
本方法采用小胤单位定量测定 NSI 索的总量,孕月brevetoxir-2 作为声素的标准品。根据小戚注射以类提取液尸的死亡时间，查出鼠羊位，并按小鼠体重，计算确定每克！肉内NSP的小鼠羊位，所测定结果代表存在于以肉内各种化学结构的NVSP：4.2试剂和材料
4.2.1丙酮（分析纯），
4.2.2二氛甲烷(分析)。
4.2. 31%湿-60生埋盐水。
4.2.4无水硫酸纳。
4.2. 5小白鼠:体重为 20 岁士1 ICR品系,娃性4.2.6称雅品：虚裸甲萨素（hrewctoxir 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花学通风柜。
4.3.5天半：诚量为0.1g。
4. 3. 6冰箱:0℃~5℃和—15℃ -—20℃ 。4.3.750011L梨形瓶或圆底烧瓶。2
4.3.8烧杯500ml.和20 ml.
4. 3. 9 布低漏斗。
4.3.15500mL抽滤瓶
4.3.11 500 mL分液漏
4.3.12玻璃珠，
4. 3. 13注射器;2 ml. 或 5 ml.为菌注射器。4.4测定步骤
4.4.1提取
SN/T 1573—2005
取样品100g川309mL丙酮均质1min。而布氏漏斗减压抽滤并收集提取浚。残留物再而样品等怀积mI丙冲洗抽滤两次，介开内酮抽滤液。沟扣滤液转移至旋转蒸发瓶净，56旋转蒸发除去内酮。将剩余水相提取物用二氛甲烷冲洗转移到5ml.的分液凝斗中，轻轻摇动,静置分层。二氯甲烷层(下层)经无水硫酸钠过滤，滤完用二氟氯甲烷继续漂洗至无水硫酸纳无色，披集洗脱液移人300ImL旋转蒸发瓶中，49℃旋转蒸发后哟存留部分即为样品脂质提取物：用1%叶温60牛坪盐水将样品脂质提取物定容牟19 rnL，添加歧璃珠荡使其悬浮混匀，所形成的悬液邸为提最液，4. 4.2小鼠试验
将小鼠称量并记录质量。每个样品注亲3只小戚：对每只试验小鼠腹腔注射」rl.提取液，同时另取只小鼠注射1 mL 1%吐温-60主理盐水作为试剂空门对照。注射讨若有1以上提取液溢出巅须将该只小鼠丢弃，并重新注汞1只小鼠：记录注射完毕时间，纸观察小鼠停止乎吸时所衰的死亡时间(到小鼠呼出最后一口止)，记录死亡间。若小鼠的中位数死亡时间小于8 ㎡ir，别要月1%吐温-69 生理盐水刘对提取逊行稀释，再注邪另一组3只小鼠,以得到大二8 nin 的死广时间。5结果讨算与报告
5. 1NSP 毒力的计算与判断
根据小暴的死广吋间,接附录A中表A，1变出相应的衔毫升注射液的小鼠单位数·质量校正系数乘以该以小淑的小鼠单位数冉飛以定窄体积,即按式(1)算,求得每克贝肉中小单位数。小風单位(Mu)/g=10M×W/100
如集小鼠的死亡时闻小于8min，就月1%吐温-60生理盐水将提取液稀释，使小鼠的死亡时间这到表A.1中死亡时闻、然后按或(2)计笋，求得衔克顶肉中NSP毒素的含量：小鼠单位(ML)/g = 10M W× D/1co式中：
M每亮升注射液的鼠单位数，(MUI/mI.)：W——重量校正系数：
D—稀释倍数.
5.2结果报告
5.2.1若小鼠的死亡时问大丁930 min.结果报告为小于 0.1 MlL./g5.2.2若小鼠的死:时间小于939 min.结果按照5.计荒得到实际结果报告。**-(2)
SN/T 1573—2095
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）
鼠单位/(MU/mL）
小鼠重量与重量校正系数的换算关系小鼠重虽/
重壁校正系数
小鼠里/
表A.2(续）
SN/T 1573—2005
重至较止系数
SN/T 1573—2005
Foreword
AnnexA of this standard is an informative annexThis standard was proposed by and is under the charge of National Regulatory Commission for certi-fication and Accreditalion.
This standard was drafted by liaoning Entry-Exit Inspection and Quarantine Bureau of the People'sRepurblic of China.
The main drafters of this standard are Zha Xin,Cao Jijuan,Qin Cheng,Song Huijun, Yu Ke and TangShouting.
This standard is a professional standard promulgated for the first time.6
SN/T 1573—2005
Inspection of neurotoxin shellfish poison in shellfishMethodof mousebiology
Scopeof Application
This Standard specifies the methods af sampling,sample preparatian and method of mouse biologyfar inspection of neurotoxin shellfish poison in shellfish anid shellfish products.This Standard is applicable to the inspection of neurotoxin shellfish poison in bivalve meat, shellfishmeat,and other edibleparts of marine shellfish.2 Terms and Definitions
This Standard adopts the following terms and definitions2.1
Neurotoxin ShellfishPaison(NsP)NsP means all marine biological taxic substances in shellfish meat, which features brevetoxin-2 forchemical constitution antd can result in nervous poison and symptom of digestive tract after being in-gestedl.
Mouse Unit (MU)
MU is defined as the minimum amount of poison required to kill a mouse weighing 20 g in 15 minwhen 1. 0 mL of shellfish extract js injected into abdominal cavity of that mouse.2.3
Median Data
Median Data means the value in the median positian when the variable values are arranged in order ofsize.
3Sampling and Sample Preparation3. 1 Inspection Lot
The quality of an inspectian lot should nat be mare than 1o0 t. The goods in the same inspection lotshall be of the same characteristics such as origin,season,specifications, packing and marks.7
SV/T 1573—2005
3. 2Sampling Quantity
According to the 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 Sampling QuantityWcight of Inspcctian Lot/t
Below 1o
51~100
3.3 Sampling Method
Sampling Point Quantity
Quantity of Mixod Samplcs
Implement random sampling according to the sampling point quantity specified in 4. 2. Cambine theoriginal samples from every 5 sampling points to form a mixed sample (determine the quantity ofcollected samples according to the type of shellfish to be inspected;ensure that the weight of inspec-ted parts is not less than 1 000 g) ,and put this mixed sample into clean container with proper markand timely deliver it to lab for inspection.3.4SamplePreparation
3. 4. 1Preparation of Test SampleThe analytic sample should be typical,i. e. select excellent shellfish from mixed samples and remavethe shell to get the meat; the meat without shell should reach 1 0D0 g,among which 200 g is to be in-spected while the reriaining Boo g is taken as saiple to be reinspected or file sarnple.If the fresh shellfish isn't inspected timely,separate the meat from the shell by use of the methodmentioned in 3. 4. 2. 1,and add shellfish meat af 200 g to HCl (0. 1 mol/L) of 100 mL, then store themeat in cold conditions (4) for inspection.3. 4. 2Preparation of Sample3.4. 2. 1 Oyster,Clam and MusselThoroughly wash outside of shellfish with clean water, cut adductor to open the shell, then rinse in-side with clean water to remove the sand and other foreign materials, Separate the meat from shellcarefully to avoid cutting or damaging body of mollusk, [t is forbidden to heat the shellfish or treat itwith anesthetic before opening the shell. Collect approximately 200 g of meat and put it in No. 10sieve,and let it drain for 5 min (avoid piling up of shellfish meat). Pick out and discard shell pieces;homogenize the shellfish meat.8
3. 4.2.2 Scallop
SN/T 1573—2005
Acquire scallop meat for inspection, the drainage and homogenization process is the same as that in3. 4. 2, 1.
3. 4. 2, 3Canned Shellfish
Transfer the entire contents (meat and liquid) of the can into homogenizer for thorough homogeniza+tion. For large can of mare than 5a0 g, drain the meat and collect all liquid, then determine theweights of meat anid volumie of liquid. Remix the solid substance and liguid according to the originalproportion andfullyhomogenizethemixturre3.4.2.4Acid-preserved ShellfishMeatDrain the acid solution from shellfish meat and fully homogenize the drained meat3. 4. 2. 5 Frozen Shellfish MeatAt the room temperature, make the frozen samples (with or without shell) semi-frozen. then openthe shell, rinse ,obtain meat and homogenize meat accorcling to the method specified in 3, 4. 2, 1,3.4. 2, 6Dried Shellfish Meat ProductsSoak the dried products in Hcl (0. 1 mol/L) solutiorn of the samte volume for 24 h-48 h at the temper-ature of 4'C , then drain and homogenize the products by use of the method in 3. 4. 2. 4.3. 5Storage of Test Sample
If the homogenized samples in 3. 4 are nat inspected tirmely.add 1ao g Hcl (10a mL.a. 1 mol/L) solu-tion to homogenized shellfish meat, then store the meat in cold conditions (4'c ) and inspect it assoon as possible.
4Method of Determination
4. 1 Method Introductionwww.bzxz.net
This methad adopts MU to determine gross amount of NsP and adopts brevetoxin-2 as the standardpoisor. Find out the MU according to the death tirne of the mouse injected by shellfish extract,anddetermine MU of NsP in shellfish meat of 1 g according to the mouse weight. The test results displayNsP of variouschemical structures inshellfishmeat.4.2Reagents and Materials
4.2. 1 Acetone (analytical pure).SN/T 1573—2005
Dichloromethane (analytical pure).1% tween 60 normal saline
Anhydrous sodium sulfate.
Mouse: weighing 20 g±1 g,ICR strain.male.4. 2. 6
Standard; brevetoxin-2
Equipment and Vessels
Rotaryevaporator.
Vacuum pump.
Homogenizer.
Chemical ventilated case.
5Balance:sensibilityreciprocal is0.1g4.3.5
Refrigerator:0℃ ~5℃ and -15℃ ~-20℃4. 3. 6
500 mL pear-shaped flask or round bottom flask.Beaker of 500 mL and beaker of 200 mL.Buchner funnel.
Suction flask of 500 mL.
Separating funnel of 500 mL.Glass bead
Injector:2 mL ar 5 mL aseptic injector.Determinatior Steps
Extract
Fetch sample af 100 g and homogenize it with acetone of 300 mL for 1 min, implement pressure re-10
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