【商检行业标准(SN)】 进出口贝类中麻痹性贝类毒素检验方法 酶链免疫吸附试验法

中华人民共和国出入境检验检疫行业标准S/1773-2006
进出口贝类中麻痹性贝类毒素检验方法酶联免疫吸附试验法
Inspectign af paralytic shelrish poisomin shelllish ror import and exportELiSA method2006-04-25发布
家数应防伪
H华和
家庭最监督检验检疫总局
2006-11-15实施
标准中国滚认证认：监督管理委员会极出并口。SN/1 1773-—20C6
公标准起意单位：中华人长共科国辽宁出人境检验冷疫局，中华人民共和国山东出人境检验检局，
本你耀牛安起草人·费际好于汉刚赵折、于灵、郑秋月、马需慈森丽、张艺本标滩系育次发布的山入境检验检疫行业标准。http://foodm
1范围
进出口贝类中麻痹性贝类毒素检验方法酶联免疫吸附试验法
SN/T 1773—2096
本标靡定广过出！压类及其制品中麻解性贝类志检验的抽样、制样和酶联免疫吸除试验检验方法，
小邻消适用于让出口表壳类贝肉、贝准和其他可食比部分的亲痹性贝类每素约筛选签测，2规范性引用文件
下列文件中的条款延过术综准的用而成为李标准条款，儿是注H期的叫用文片，其殖后所有的修改的（个包洁期课的内）或餐订版均不道币本标维，然而，設贴根沿标准达成协设的各方严究是否可使用这些文件的最就版术。儿是不注日其的于月文件，其最新版本适月十太标准GB/T6682分析实验室用水规格和实验方3术语和定文
下列术语定义活用下本标准。
麻痹性页类毒素Paralyticsleilrislpuisoaing.P'sr化学结构以右房始毒素（Sxox:为代丧的，我食后可产牛廉疗作用的存在一贝类体内的海生物幸性物质的总称，
4抽样和制样
4.1检验批
以不超度1C为检验批。可一格验法的询品度有州尚图特，如包装标理、他、季节和现恪等。
4.2抽栏数量
接各检验批的数量·依表1拍收栏品：表1
检验批的数型/
0以下
51--109
刘为散要的应均匀样。
4.3抛群方法
油样点(件)妝
浩证效
按1.2规证的抛样点（件)数.随机拖收样站。衔5点（件）拆取的真始详品组成“个混合郑：即检验产品）。其质量不少示2kg.人清站签器内，.时标记汽，改时进实验至检验4.4分析栏品的采朱
分析样品婴有分药代友性·依受检以类儿钟决定品的采集会，取群个数不少二12个员美会htt
SN/T 1773 --2006
休，即从？呢栏品中城选良好的贝来恶，志竞内量应达℃。对个体划小的品，则以充后齿率不少+230 鸟末爽定采集个数。新鲜贝类不能及送检，按4.5.1方法将贝肉分离，将泌水后的20%g贝肉放人100折L整酸(.1mml/.),」冷磁保切勿冷冻），格检。4.5试群削备
4. 5. 牡蛎、烩及贻贝
用清水将贝光外衣彻吸洗净，切断闭弱起，北壳，也更蜜水淋肉部大除视沙及其他外来物。格闭壳肌和造接在胶合部的维综分并，存红最蛋具肉优物割破肉偿，并前不要加热或用麻醉。收集约2C0g肉置丁饰，中求不套使肉地积，格出醇壳拿杂将县肉均质，4.5.2扇贝
瑕可食部分用作检测：满下及均质过程间 4.，1.4.5.3贝类罐头
将镶内所有内容物（肉及液体)例人均质器氛芬均质好妇果是大擎，将只肉源水并收集沥下的液体，别称恒·将同形物和汤注按比例门脆芬将质。4.5. 4用酸保存的贝肉
去峻液·分别存效贝图凝敏微凝沥个的贝闪充分均贞，4.5.5冷冻灵类
在品下，良冷冻的样追我范戴脱壳的星冷冻状态技核劳法无光、淋洗玫内、乌质，4. 6 试样保存
上述4.5中经为质滤理静择战婚不能及尚橙测市最准00包质贝肉加人100m，盐酸（0.1no./1.)溶液，官于11冷藏保存（尽可能发时检验）5测定方法
5.1原理
术方法能测定世健是帝争经联免安吸附试验度应高麻性换类育紫湾生区类南囊酶标物亲争床兴性贝美与素航探同博释班性类款素抗述与指建钵遥接。没有被结合的醇标记物在选落步聚被除大。结食销比物将元包的发爸就转化为然包产物而人反应停山液后化颜色出蓝转变为首色，让4心避液长的解际仪测量微能落液的吸光度值中的麻裤性贝类每素溶液！吸光度惯成反比，按绘制模靠围线完呈计算5.2试剂和材料
除另有规定处，所有花掌剂均为分析或牵花试剂，尽为按6632现定的一级水，5. 2. 0. [ mol/z 书
5. 2. 2 3 mo/L 热取.
5.2.3半对效坐标纸，
5.2.4标准动质液约被：经酸香特净的艺惊作为保境液冷载时，无限期德笠。5.2.5标记物浓疫。
5.2.6抗体液缩液。
5.2.7基质，发包剂，
5.2.8疫应终止液：1 mol/L硫鞍，5.2.9级冲液
5.2.10商业化试剂盒测定低报（或满足因内外限量要求）回收率达到本标泄的要求，照起适用于本合品伙伴网
5.3仪器和设备
5.3.1微孔板标议（450m）
5.3.2均贴器，
5. 3. 3等心析
5.3.4微量样器.o m,e μLoo L
5.3.5微量多通加样器33uL，1290L5.4试样的提取和净裙bZxz.net
SN/T 1773--200
称皮10.15试杆（精效人酸落如乘洋品为较于操的贝肉，加人140 ml0.1mal/：盐说落液播家数等同者部齐搅拌mm00xg离心10min，离心后控制pII仙，用=mo1/=盐酸浴液制节到4.0以下，取100正清微带绿缓冲液较1：10稀整（1+3)，取0江按53条边行联免疫测定，此时约粘释借数高城度的栏品，超出标准由线范国，可迟步用凝冲液蒂释，直至样战浓度在标推出践葱以波家5.5醇联免癌吸驸试验的测定
将足够数呈的微孔杀摘人微班疆标解和辩液分别做复孔），起录标阵滋和样的置。加人6个适当的痹质类荐素标准教译额释品到各微孔，加人0u麻牌性员关素酶标记物至旬个微孔，轻轻混合，再人痘性贝类克素护体，彻底混合粘胶纸封在微孔以防溶液祥发，2（:~25℃黑倍避光处孵脊将微乳巢谢赔在破水纸换打数欲，以保证完全除去微孔中的液体，专个微孔注满重然留水洲洗后损通重复以上院板操推板铁发ICCtL某质/发色试剂签每个微孔，轻拍湿幻.25℃熙情光处我然10发应终止液至每个微孔中，轻拍得约片，以空自谢，直并记录新个徽溶液50nm波长的吸光度值。5,6结果的计算和表述
5.6.1计算百分比吸光度值
计箕麻穿性贝类态标准液和税被的平均吸光度值接和样液的店分比极光度：
A——吸为医值的自
穿求得每个麻痹性贝类毒素标谁液..(1)
S5个适当的麻癫拌类荐素标谥被战样较韵平均吸光度s——0u/ku的麻解顺类益素标维液乎萄吸光度值。5.6.2绘制校正曲线
米效为拟坐标以麻贝美可索滑最装搜（g/kg）（对微级）为精尘标，绘以百分出吸光度仿（菀
而出戚疾性贝为克素标准液音添嫂要好麻练性贝类英囊溶液限度好校正綫。在次试验均应乐新瓷制校上恒线。
5.6.3结是的计算
在562绘制的度正问裁上读取推波口分比光度试所对应的棘痹性贝类毒豪欲度即必试些中麻疗性以类索的含量（/k）。为获衔样品中的案性以举毒的实弥浓衰（/)，从校正而线上读出的浓度值必须乘以相对应约器释系数，5.5.4结累的表述
当测定值小于5c/k时则报当媒痹性贝类素的含量为小于5/kg。当测宗俏六于等下30 g/kg对,则报件麻性贝类毒索的实际测定俏，合品伙伴网h：
SN/T 1773-—2006
6测定低限.回敢率
6.1 测定低限
本方法的视实低限为53 /k、
6.2回收率
本方法的收率约为的与。
健康和安全
征们原痹性测类克素含量俏大于909g/kg示惯例表示方式为80g/c0g的群品即被认为是有害的对人食历不安念
标准液含位麻察性可类老紊，成裁别小心，避绝接触。减终止滋为1.硫酸，避免落触克肤。http:/
Foreword
SN/1 1773—2006
Tais Standard wes proposed by and is under the charge of the Certification and Accreditation Administratian of the People's Republic of China.This Standardl was released upon approval of the General Administration of Quality Supervision, In-spactior aracl Quarantine uf the Peapte's Republic uf China.This Standarcd was drafted by Lioaning Entry-Exit Inspection and Quairantine Bureau of the Penple'sRepublic uf Chirna arid Shandong Entry-Exit Inspection& Quarartine Bureal of the People's Republic olChina.
This Stanbard was major drafted by Cao Jijuan, Yu Bing, Wang Cang. Zhao Xin, Yu Ling- Zheng Qiuyuo, Ma Huirui, LiLi、and Zhang Yibing.This Standard is promL.Igcted for tie first tme.全品伙伴网
SN/T1773—2006
inspection of paralytic shellfish poisonin shellfish for import and export-ELiSA methodScope
This Standard specities thetard ELISA for the deterrrina-tion of paralytic shellfish poison tr shelffsh' ara fts pracrurcts tak arnpart and export.This Standard is applicanle to the determinatign.foaray
ligament and otrcr edible parts of bivalve tor tmpe2 Narmative references
snellfish oisor in molluse meat, shelCeno axport
Thefollowing standardscohtatngprovisions.,which are.used.in.this text for reference.constitutcprovisions of this Standara For datec references subseguent amiendmenls (exclude correction! toor revisions of any or these pusticationsi shat ingt erplyutbathis standard. All parties are subject toagrecmonts based on this standard are eneulriged ru myastigate the possicility of applying the mostrecent ecitions of the stanclarcis indicated below. For undated references the latesl editiuri of thepublication referred lo appias.GB/T6682Waler[or analyticallatiaratory use3
Terms and definition?
The following terms art
Paralytic shellfish poisoi
tost methods
finitionsare appicas
etothisStandafe
Parstytic Shellfist Poisoning, PSPTypical chemical constitution is that of.dtoxin.aqeneialterm
ftoxic s.bstance in halobios sucias snellfish. which will conseguentlyeause.paralysis in hurhah bethgs who et the srellfish.4Samplingandsamplepreparatian4. 1 Inspectlon lot
No more than 1co t of commodities are sampled as one inspection lot.anc! te same lot of commad:.6
ht
SN/T 1773- -2006
lies shicuic have the same characteristics such as packing, referencc standarrls: origin. season andspecificetion.
4.2 Sampling quantity
Sanple as follows aacording to the cammocity quantity in cach inspcctiar lot ,sec Tablc1.Weight or inssectiort jotst
ssamapingnmbar
Tewarthun1n
5: --100
Uniform sampl'ng in case of bulk package:4. 3Sampling method
Kumter of mixcc: samples
Randomly sample accorditgf tathe nurnbetstipulated in 4-2.: Take jaut:one from each 5 samples andmix tlhem into mixed sanplesey inspectan sangplesihwelghtnotess than 2 kg. Put -he samplesin a c ean container, seslana markiteand then send it.to taelab.timely for inspectior.1
4.4CollectionofamalyticalsamplesThe aralytical samoles should be typical ones, and its number clepelnids on the species of shellfish.The sampling nurnber shouild rot ketess than 12j.eselect good: shellfish from 2 ky :nixed sarnples.aa:l renoye the shell maasr
la wcigh 2co at least Far.too smail shellfish, the sampies shauldbe cullectedl as loly as ghe theaswithout shalis waigks no less than 200 g at least.It the fresh shelltish cannot.be'sant to inspect th time, seperatermeat ttom shell acconding to 4. 5. 1 :put 2cog rneat withaut xcessive water in.ibo mtHcl (0. 1 rrol/L2Sentsture it in a cool place al 4℃(freezing is forbiddefoemspcctian. Sample preparatian
Oyster, clam and.m?
Well clear: the exlerior of shellsWthi ctesn water. cut afkadductor mLscle. open the shells. scouroff the mud. sanc and otner foreign matters inside with heavy distilled water. Separate tae adduciormuscle fram other oarts,carefully take ouit the meat wvitinotrt lacerat ng it. Dn nnt icnt or isn anes.thetic before opening the shels. Put auoul 200g rneat in H screerl lo drain excessive wailer lor 5:nin(cispcrsc mcat uniformly): anri pick out broken shells and othcr fareign matters. and homogenizethe eat.
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Sx/T773--2006
1. 5. 2Scallop
Inspect the edible part. The procasses ol draining and homogenization are as same as specitied in4.5.1.
4.5.3Canned shellfish
Put ali the supstances (ineal and liquid) in the container into honogenizer for fu: homogenization. Ifthe container is big, weigh the drained excessive water and meat respectively, anu rnix the su'd aridliquid i:1 proportion (1 : 1) and homogenize them fully.Storingmeat in acid
Drain acid licLior, store meat and acid liquor separately, arad fufly ho'nogenize meat without exces-sive water.
Freezing shellfish
At room temperature: make the Frozen sarnp'es (with or withaut shells) in semi-frozen state. ardthen opa the shells, clcan. take out meat and homogenize it in the processes of 4. 5.1.4.6Preserving samples
If the homogenized samples in 4. 5 cannot be inspected in time, store 100 g homcgenizad imveat 'n100 mL HCl (0. 1 mol/L) at 4'C (inspect it as soon as possible).Determination method
5.7 Prineipie
This deternination method is based on competing ELisA reactio. Fref paralytic sheilf sh poisoncompctos w'th paralytic shcllfish poison enzyme-lapeled substarices fur paralytic shellfish puisun an:Lbuuly, arid al 'the same zime paralytic shellfish poison antibocly is linkod w th capturo cntibody. Theenzyme-labe ed substances tnat are 'ot linkec with others ere removed in the process of washing.The linked enzyme-labeled substanccs will convcrt colorless chro:nogen c reagent into blue prod.ict.Afler addirag reaction stop soiution. the blle tuitns yellow. Use lab-systernis dragon in 45(nm wavc.lcngth to mcasuro the absorption value of so:ution in wells. the para.ylie shel Fish poison solition insample is inversely proportional to the absorption valun and caleulate aceording to the drawn calibra-tiori curve.
E. 2 Reagent and material
SV/T 1773—2006
All cherrical reagjents are divided into aralytrcel reagents or biochemical reagents unless otherwisespecified,Watorshould be class-Awater as specified inGB/T66825. 2. 1 0. 1 mol/E HCI
5.2.2 5mel/L HCl
5. 2.3 Semilogarithmic paper.5. 2.4 Concentrated solution of slarivarc substance: through acicificatian, inclucding 20% etinanof asprotectivesolu.t:on.permanentlystable in casecaf cold storage5. 2.5
Corcentratecl solution ol enzyrne labeled substances.Concertrated solution ot antibody.Strurme/chromogenic reagent.5,2.8
Reaction stop solution:1 ritl/LHsa.5. 2.9
Buffer solution.
5.2. 10 Corrnercial kit that satisfics the requirerents in this Stanciarc in lower lirnit cletermination(or satisfiestch reqajirements for limits at home and abroad) and recovery perccnt s also ajpplicablethis Standarri.
5.3Instrument and equipment
5.3.1 Microplate lab-systems dragon (450nm),5,3.2
Hornogenizer:
Centrifugal rachine.
Micra aop:icetor:50 uL.100 mL,500 μrl.5. 3. 5 Multi-channel micro applicator: 50 μL, 100 μL.5. 4 Exracion and purification of samplesWcigh accurately 10. 0 g samples ( acurate in 0. 1 g). acld 70 mL 0. 1 mo./L HCl solutior (if sample is5
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SV/T1773—2006
reletively dry meat, accl 1voml o. 1 mal/f. Hcl solution in thc same coefficient of diliticn. boil atidmix thern for 5 rmin: decerler 3 500 y sucll sarmple at 4'c for 10 min, ans t:ner cartrol ph valic: uso5 mol/L HCF solution to adjust to bhc iower than 4. C: take 1Co μl cleer liquid and dilule it with buffetsolutio' in the dilution ralio of 1 : 10 (1 -+ 9). andl then take 50 u for ELiSA according to 5. b. Theextensior rate this time is Bo, For high-concentration samiple, if beyorct the tange o' stancard curve.furthire diute il w th ouller solution tmtil its concentratinn is within thc range.5. 5Determination af ELISA:
Insert adegLate ELISA plates n the midropore frame chake muitipporesgespectively for ller andsample solution). and record'the posit ons of tliter aincf samalgsotution ad 6 portions of appropri.ate paralytic shellftish po:son titer and samaln solution :ntaveryrmciapore (50 μL in each one), add5o :L paralytic shellfish poison enzyme laueled substancesto every micropore and then mix themgently; aftcrwards.add 5c μL para ytic shellfish poisonantibocly into the mixedl soiution and mixthen fully; seal the microjore with viscosepaperifor feer of volatilization of solution.ard incuoateit in a dark piace at 2u'c ~ 25 for 1aniingbottbirytis micropore frame enove absorbent peper andllap it several tines so as to ahsalutelyeliminate solutian in micropores: fill every rricropure withheavy distlled for rinsingl and theneflap the Jrame until no zolutign remairedi. tepeat the asoveprocesses 5 times; add 1on ul stromi/chramnogenie reagent in, everymleropore: lightly flap it tomakeitwellmixedand theninaubeteftinadark placeat20t25etori5bin:add 100μL reactionstop solution in evcry microbare lighily.flaptt enaket wentee set ernpty tube ta zero.meastlre and recorci the absorpiian value of solutiori (45(' nm wavelength) in every micropore.5, 6 Calculation and formulation of results5. 6. 1Absorption porccntageCalculate thc averagc absorption values of titer and sample.solt.tiun twork oul thepercentabsorption vaitaeofeach portialfish poisan according to formuitaxWhere
percent absorpt:on'yalue
orandsam
S--.. average absorption valae of e-portigns of-aporoprialepe?sample solution:
akitc she ifish poison. anclotion of paralytic shell.
shellrish paiscn titer and
S,--nvcrage absorption value af es/kg paralytic shellfish porsor titer.Plotting calibraticn curve
Percent absorption values (arithnetic degree) s Y courdinate, co:centration μg/kg) (logarithnmcegree) ot paralytic shellfish poison sn ution as x coord'nate. plot the calioraliui curve fur ilie μer-cent absurption valule of paralylic shellrish poison titer anc the concertretion of paralytic shellfishht
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