SN T 1996-2007 贝类中腹泻性贝类毒素检验方法酶联免疫吸附法.pdf

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1、中华人民共和国出入境检验检疫行业标准SN/T 1996-2007 贝类中腹泻性贝类毒素检验方法酶联免疫吸附法Determination of diarrhetic shellfish poison in shellfish一ELISA method 2007-12-24发布2008-07-01实施白宫tiJ良品坠Abjp吕旦发布标准搜搜网各类标准行业资料免费下载前盲小勾i、f由国家认tlE认口f1监督管理受员会，是出-归U本东准起巾.单位:中华人民只相|吨辽宁陆人拢字号价检疫局，本东准T飞雪耳边币人:千兵、曹li在娟.夜1甘?光、，臂、丽JJ.东t， 射琐、李丽.孙必4伞klt;在东ilt，发

2、布的金验检疫fi业标准.标准拨拨网阳飞饥各类标准行业资料免费下载S-VT 1996-2007 1 范围贝类申腹泻性贝类毒素检验方法酶联免疫眼附法伞klt;在KIl述了极泻忡员类#素金验的抽样、副祥和10制提f:r.i交检验力-t去.本东准适用十世手产以71t类可r:r期尚分的，IW:肖性lyl类毒素的字号段、2 规范性引用文件F乒州u义件干l巾l卡t的条U以u通匾过，乍1仁手斗斗f;仇V咱咖川l扣、J;准挥自忏的;斗j让训l用11肌11比州1;l成戊为+伞札，邻tS.vT 1996-2007 自的斗t修善改币(1个4包J阳;!il也u山误自的斗l川，叮才结容手)y-吹且修f际￥钊t汀丁版i均坦

3、叶私札J立.淫i温唾门于斗水.t.f准悻.然.，奴胁根据非心J1:萨达J正协议的手斗力矿光是台I使用这辈义;件的最新版-t，:凡是个i主U剔的11m义件.具;妻麦新版非.用于水如位动SI (!2 iSI 化学，.i构以六口牧海绵自主(okadaicu:d，吁j称()A):业其汀1:I结为代衷的，rA(tJri可户1:J. ij;:!沟沟宁要特亚的(1&十)1尖j本内的:海洋牛防毒性物质的总.iT- 3.2 缩略i吉3.2. 1 DSP diarrhetic shellfish IOiIlSOIl 腹泻险IJH主毒R3.2.2 I!LlSA en巧melinked mmunl.lsorbenl

4、:l部ay院联免咬l剧精前段，4 抽样和制样参照51C2!H J叫志Jl;tli5 测定方法5. 1 测定原理本方法!I定基础是觅争仨院联.疫!王成j:(离的腹泻性l人类喜R与1主肖性)1尖毒两标i;勿必争.叹:写件仄类忐东抗体.没仔被J吉兮的自每杠、记衫:nn!:i在步骤Q1夜|涂手是冬3每，)道白显色汗11如|人正1j:11IUH1f.育.声介的t轨i、i三物将.A:色的发色jjI!转化为监色的产物.川人反应终.Ji:.1i主rjftJ!颜色由监转变为贵色。了1:I J l:m &:长的隐斗、仪现II蚊子L滚:寰的吸1t度值.佯品11的夜污仲贝类存在济11宜与吸光，专值成j豆比.1发绘制的

5、ti.u.:曲线定量汁:究45.2 试剂和材料除另有u定外，所有他学试剂均为分祈纯.d为可走茶馆iJ;. 标准拨拨网阳飞饥各类标准行业资料免费下载stl 1996-2007 5.2. 1 )O只甲醉济;在5.2.2 半对在自坐问.纸5.2.3 DSI标在物质45.2. 4 h;t二物.5./.5 包饺J.泻fl:K类毒素抗体的:孔板5./.6 以!贡O:t氧也康奈发P=.)IJ( 1叫甲足联与t.Q主人5.2.7 反对终11.浓:1IlwVL.定自古.5.2.8 商业化试汗1:盒拌;r低限fU口11失率达主IJ木.t，.、j佳的要求rJl!.j适兮于本杠、准e5. 3 仪摆相设备5.3. 1

6、h;义。5.3./ J斗攻击番。5.3. 3 !心组:4(; .:; O()(;e 5.3.4 微量加付器:50rJ.L，、5.3.5 微E多iiji，重如I佯帮:J d.;! I CJ d.; 5.4 试样的提取和净化和;1京lj.(.1 g L!:佯(而确斗j. 1 g) .加入;，-11 9(.1 .巾回事容tf汪.1坦:改11:1ill ,., Z 1111 r_. H.: (.1反两心lCm:n. t甘心l取卡肯浪.同军崇他l.1, ?!在I 2稀f扣1+l、取;CtI.T. ?!豆、J进行前联免疫1i!J:t. jft .I问稀薄1肯放是10.白浓度的H!lt，超出.t，i1(曲线

7、忧国.口l成-步用以t;.气甲醉j归宿苟样，直们测定伯布.t，;(优曲线范围!.I;5.5 酶联免疫测定1号足够数串的悦宁L条插入.x孔架f如:准液和|样被:.l.I)IJ:复孔3.记录如1:在液和l佯浪IfjFi丘。别人。11.2个、，个腹泻忡贝类毒素f;、准放军l佯tf注:IJ;各自的.x孔.每1、标准液和1年i夜应史刀新的吸头.加入、),t 1，腹i雪1生顶尖孬实酶东:-:4均至4叶个微孔、迅速究分混合.22C ，.，2、儿:避元处弹?自10r:1in , :向微孔当I纠旨在l吸水纸u川lL盯大，以保:f.究气写了|涂1.;.放孔中(t(J液体，fL个微孔沁人250;.tL 先液;中狱后

8、lil干.丐丰以上ic板:袅.f午1次别人5(J，1.绞色ifl年每个微;11.11 ，轻拍?月二J.2c.;ica市4哇!护士处)J手育(i min. JIU久CILL反;.)l终11:液至4号个激化中.j理没生JJ号，f主1:)min内以们气只r;-;向调了苓测量社记录每个侬孔i容浪I:;:inm i皮i汇的吸光度且.5.6 结果的计算和表淫5.6. 1 计算百分比眼光度值H贷|诙泻忡归炎毒素.tji1(浓利TH液。(Jf.均l贬光度白，:式(1)分别JEi事1:j个l诙泻忡归炎毒素.tji1(浪和己LJf1宜的内分t二吸1t;专值2，1一主/lC O?; A中:A一-ri公比吸光度刊腹泻

9、1生顶头毒素标准液戎样i夜的平均吸也)副主a队一-:)I!g-! L (t(J I诙泻卡归尖毒素4ltJ优浓(t(Jf.均l贬光1支伯5.6.2 绘制标准副主是( 1 ) 以百分眈l没光!在(ti(伊术夜).I.从$标.以I!J.j五性1)尖.浓度(J缸k旦川对数级).ItP时示绘制向股市性民+导京东:雀液百分比吸元1划由主j肢得住呗尖寿家浓度的标准曲线，4-t;欠试1沪均j. 注意，研绘制标往由主L5.6.3 结果的计算了1:巴，3.2绘制的问.j传llJli戈J.佯i夜nJttl:吸77lt度值民r，:t)主的版污仲贝类存款浓度jJ为试佯1ij写.、标准拨拨网阳飞饥各类标准行业资料免费下载

10、.f:贝在毒素市呈(，ug/kg)5.6.4 结果的表迷当划定值小于lJIl,l;/kg :寸.!;IH主fiT腹泻仲项奖存东含亘小于10/俏峙，当;!II定(ti六十每每十l!,g/ kg 1., 1!Hli;!;实际;!II定数:、6 U!l1定低限6. 1 测定低阪木1i法问JI:;t 1rJ:限为IC :.Lg/kg, 6./ 回收率7 健康和云盒S,VT 1996-2007 11:何腹泻悻贝类必寻Jf守主值大于1h ，I!日!I (JR.2飞)，I!日!I (J飞)目的惮品II被认为是J-j赛的对人类(.t闸不安全4标准浪含有.t￥仨I!类毒.!也特别小.(:.t1，也接做4反应终止

11、以为1m:二1.硫酸.滥免i豆制度快.标准拨拨网阳飞饥各类标准行业资料免费下载、.stl 1996-2007 Foreword This St口nd口rdwns proposed hyand is IInder the charge of the National Reglllatory Commission for Certification and Accreditation. This Standard was drafted by Lioaning Entry-Exit Inspection and Quarantine Bureau of the Peoples Republic o

12、f China and Dandong Entry-Exit Inspection and Quarantine Bureau. This Stand自rdw自srn自inlydrarted by Yu Bing Cao Jijuan Gao Shiguang,Ma Lidan,Jin Dongqu自nXieYan. Li Li日ndSun Ji巳This Stnnd口rdw口spromulgnted for the first time. 标准拨拨网阳飞饥各类标准行业资料免费下载S.vT 1996-2007 Determination of diarrhetic shellfish pois

13、on in shellfish一ELlSA method Scope This stand口rdsper. ifies the methods of sampling sample preparation and ELlSA fm the determina tion of diarrhetic shellfish poison. This standard is applicable to the determ ination of diarrhetic shellfish poison in molluse ll1eat , shell liJall1ent and other edibl

14、e parts of bivalve for ill1port and export. Positive resLllts ll1ust be confi nned by orther methods. 2 I唱。rmativereferences The following stnndnrds r.ontl in provisions、whir.hare lIsed in this text for referenr.e、r.onstituteprovisions of this Standard. For dated references. subsequent amendments (e

15、xcluding corrections) or revisions of any of these publications shall not apply to this Standard. AII parties subject to a!ree ll1ents based on this Standard are encouraged to investigate the possibi lity of applying the 1l10st re cent editions of the standards indicated below. For undated reference

16、s the latest editions of the pub lications rererred to are applicable. SN 0294 Method for the deterrnination of diarrhetic shellfish poison in shellfi!:h for irnport日ndexport 3 Terms .definitions and abbreviation 3. 1 Terms and definition The following tenns and definitions are appli臼bleto this Stan

17、dard. 3. 1.1 Diarrhetic shell rish poison, DSP Typi臼Ichernical constitution!:日retho!:e of okadaic acid and its rarnific日tions.a general terrn of tox i r. sllhstances i n ha loh ios sllch ilS shellf ish、whichwill conseqLJentlyuse d iarrhen in hLJmiln he ings who eat the shellfish. 口标准拨拨网阳飞饥各类标准行业资料免费

18、下载stl 1996-2007 3. 2 abbreviation 3.2.1 Diarrhetic !ihellfish poinson. DSP. 3.2.2 Enzyme Linked Irnrnunosorbent Assay ELlSA. 4 Sampling and sample preparation Irnplementation i!i !iubject to sampling and !iarnple prep日ration!itandard method!i in SN 0294. 5 Determination method 5. 1 Principle This de

19、termination method is based on competing ELlSA reaction. Free diarrhetic shellfish poison cornpetes with enzyme-Iabeled diarrhetic shellfi!:h poison sub!itance for diarrhetic shellfi!:h poi!ion an tibody. The lln-linked enzyme-Iabeled substance is removed in the process of washinj. The enzyme stroma

20、 and chromogenic reagent are added into the well and incubated. The linked enzyme-Iabeled subst日ncethen convert!: colorless chrornogenic reagent into blue product. which will turn to yellow after adding reaction stop solution. Measure the absorption v自lueof solution in wells in 450nm w自velength and

21、calculate the concentration of the diarrhetic shellfish poison in sample according to the drawn c日librationcurve. 5.2 Reagents ;mr.l mlterials AII chemical reagents shoLlld be analytical reagents unless otherwise specified.叭latershould be di distilled. 5.2.1 90% Methyl alcohol sol Lltion. 5.2.2 Semi

22、logarithrnic paper. 5.2.3 DSP st日ndard!iubstanc巳5. 2. 4 Enzvme-Iabe led substance. 5.2.5 Diarrhetic shellfish poison antibodv coated micropore plate. 5.2.6 Sustrate (urea peroxide)/chrornojenic reagent (tetramethylbenzidine). 5.2.7 Reaction stop solution, 1 mol/L H,SO. 民标准拨拨网阳飞饥各类标准行业资料免费下载S.vT 1996

23、-2007 5.2.8 Commercial kit that satisfies the requirements of determination limit and recovery in this Standnrd is nlso口pplir.口ble.5. 3 1 nst rLlll1ents and equ i pments 5.3.1 Enzyme-linked amlyzer. 5.3.2 HomoJenizer. 5.3.3 Centrifllger:4C 3000g. 5.3.4 Micro applicator: 50 I1L 5.3.5 Mlllti-chnnnel m

24、icro applicator: 50 ,u L. 100 ,.lL 5.4 Extraction and purification of salple Weigh 10.0 9 sample (jmprecision, 0.1 g). then ldd 5 times weight of 90% methyl l lcohol sollJtion. Hornojenize the mixture for 1 min2 min. Centriruge a1 4C , 3 000 9 for 10 min then t rans f e r the supernatant into a new

25、vial and dilute with didistilled water in ratio of 1 : 2 (1 -1). Remove 50 I1L of dilu1ion for ELlSA according to 5. 5. By now the extension rate is 10. For high-concentration sample whose determination reslllt exceer:ls the range of standard Cllrve, fllrther dillltion sholllr:l be done with 90% rne

26、thyl alcohol !iolution until the concentration i!: within the ranje. 5.5 Determination of ELlSA In!:ert adequate ELlSA !:trip!: into the rnicropore frame (double test!i for !:tandard and !:ample !iolu tions respectively) and record their positions. Add 2 10 5 concentrations of standard solutions as

27、well as sample sollltions of diarrhetic shellfish poison into different micropore (50L each) using new tips. Then ldd 50 j.L enzyme-Ilbeler:l dilrrhetic shellfish poison sllbstlnce into every micropore and mix surriciently and quickly. Incubate the mixtLI res in dark at 22C 25C for 10 min. Bottom II

28、p the frame on a piece of absorbent paper and flap it for several till1es so as 10 absolutely relove the liquid in the micropores. Wash the micropores with 250L washin! solution each and flap the frame until no liqlJid reml ined in the micropores. Repelt 1he above washing step for 4 times. Add 50 j.

29、L chromogenic reagent into every rnicropore and mix well by light flap!:. Incubate the mixture in dark at 22C 25C for 6 ll1in. Add 50 I1L reaction stop solution into every ll1icropore and mix well rapid Iy. Measllre in 450 nm wavelength and record the absorption value of solution in every micropore

30、af ter setting the vnllle of the empty micropore to zem fm 10 min. 5.6 CalcLl lation and Expression of reslllts 5.6.1 Calculation of the ahsorption percentlge Calculate the averaJe absorption valLles of diarrhetic shellfish poison standards and samples.叭lork标准拨拨网阳飞饥各类标准行业资料免费下载stl 1996-2007 out the

31、percent absorption value of each standard and sample according to Formul自(1): A=丢x1肌( 1 ) where A percent absorption value; S-average ab!iorption value of diarrhetic !ihellfish poison !itandard or sample: Sc averlge ahsorption vl llle of 0 :g/L r.l iarrhetic shellfish poison stnnd口时.5.6. 2 PlottinJ

32、standard cLlrve By setting the percent ab!:orption values and the logarithrn concentration values of the diarrhetic shellfish poison stand口rds(j.g!kg) , plot the stlnr.lnrr.l Cllrve fm percent absorption vallle and concen tration of diarrhetic shellfish poison. Plotting should be done for every expe

33、riment. 5.6.3 Calculation of result The diarrhetic !:hellfi!ih poi!ion concentration( /lg !kg of !:arnple臼nbe calculated according to it!: per cent lbsorption Vl llle lnr:l the standarr:l cllrve preciser.l in 5.6.2. 5.6.4 Expression of result 叭Ihenthe test result is、，10/,g/kg.the concentration of di

34、arrhetic shellfi!:h poison should be re(Jor ted a!:、10/lg !kg. When the test result is注10坤/kg.the concentration of d iarrhetic shellfish poison should be repor ted according to the exact value. 6 Determination limit and recovery 6. 1 Determination limit The determination limit or this method is 10 :

35、唱ikg.6. 2 Rer.ove ry The recovery of this method is 85% -90%. 7 Health and safety Any sample whose concentration of diarrhetic shellfish poison above 16 lg/100 9-20 lg/100 9 is considered to be harmful and Llnsafe for human consumption. 8 标准拨拨网阳飞饥各类标准行业资料免费下载S.vT 1996-2007 Treat the standard solutio

36、ns carefully and avoid contact since they contains diarrhetic shellfish poi son. Avoid skin contact with the reaction stop solution which is 1 molL H2SO们标准拨拨网阳飞饥各类标准行业资料免费下载, 、khOON-白白FHZm中华人民共和国出入境检验检疫行业标准贝类中腹泻性贝类毒素检验方法酶联免疫吸附j去SN / T 1996- 2007 可k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:68523946685 17548 中国标准出版社秦皇岛印刷厂印刷兴印张1字数15千字2008年3月第一次印刷兴书号:15 5066 . 2-1845 6 标准搜搜网WrM肌bzsoso.corn各类标准行业资料免费下载定价10.00元2008年3月第二版印数1-2000 1/ 16 开本880X1230 SN/ T 1996-2007