【商检行业标准(SN)】 贝类中腹泻性贝类毒素检验方法 酶连免疫吸附法

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-ELISA method
2007-12-24发布
中华人民共和国
国家质量监督检验检疫总局
2008-07-01实施
本标准由国家认证认可监督管理委员会是出归口本标起草单位：中华人民共和国辽宁出人境检验检这局，本标准亡安冠节人：于乓,曹际娟高世光,麻丽小金东议、谢毯李丽,孙然本标准系次发布的验验检疫行业标准S>/T1996—2007
1范围
贝类中腹泻性贝类毒素检验方法酶联免疫吸附法
标准规定了腹泻性贝类毒素脸验的油样、制样利嗨联免检验方法。本标准适用于海产效光类可食用部分的腹泻性贝类毒索的检验。2规范性引用文件
S>/T 1996—2007
下划文件小的蔡感通冠不你滩的而匝成本弥滩的亲歌，凡是注口期的而文件·随后所有的修改（不包括助误的内穿)或修订版均不适门于本长准·熟而，最励根据本标准达成协设的各方矿究是否叫使这些文性的最新版本凡是不注口期的川文件·具最新版适而于不标雅，SN0291出11贝类腹污贝类毒素检验力法3定义、术语和缩略语
3. 1定义和术语
下列术语利是义适而于本你滩。3.1.1
腹泻性贝类毒素diarrhelic shellfish puison.lsT化学结构以大口软海绵酸（kidaicl：简称A)及其衍牛物为代表的：摄食后可产牛以腹泻为主安特征的存在十类体内的海洋牛物毒性物质的总称。3.2缩略语
Dspdiarrhetic shellfish poinson腹泻性贝类毒系。
ELISA enzyme linked immunosorbent ussay酶联免疫设附试验，
4抽样和制样
参照SN（294舰定执行
5测定方法
5.1测定原理
本方法的测定基础是竞争性酶联免疫反成、游离的腹泻性！类毒系与孩泻性从美毒索酶标记物兜并换泻性贝类持素抗优，没在被结会的酶板记物在沈涤步骤服除去：将嗨基质显色剂加人到孔中准Ⅱ孵育·结分的酶小证物将儿色闷发色剂转化为蓝色的产物·剂人及应终正液店颜色由蓝转变为黄色。在15rm波长的嗨尔仪测量徽孔溶液的吸光度值：样品中的拔泻性贝类毒素溶液与吸光妄值反比：按绘制的较止曲线定量计笋5.2试剂和材料
除另有规定外，所有化学试剂均为分析纯：水为重裘馏水。-
$N/T1996—2007
5.2.190次甲醇济液
5.2.2半对数坐标纸
5.2.3LSP标准物质
酶标记物：
5.2.5包被腹泻性贝类毒素抗体的微孔板5.2.6
案质(过氧化尿素)发作剂（四甲其联苯胺）。5.2.7
反市终液:1 ol/L硫酸，
商业化试剂盒益测定低限和可收率达到本标准的要求则适今于标准。5.3仪器和设备
5.3.1标仪。
5.3.2均度器。
5.3.3离心机：4.30cg
5.3.4微量加样器:50μ
5.3.5微量多通道加样器：50.1005.4试样的提取和净化
称10.诚样（精确至0.1）.加人可倍9巾醇溶液均页1min~2ir.3g离心l:m离心后琅上清液·月重蒸馏水妾！：2稀释1十I）琅iul获进行碍联免疫测定。此时的稀释倍数是1，官浓度的样品，如超出标准曲绒范围可进，步用法甲醇济液稀释，直半测定伯在标准曲线范围双
5.5酶联免疫测定
将足够数量的微乱条插人微孔架标谁波利样液分别敬复孔）记录标谁液和样液的位丘。孔人点.2个个腹泻性贝类毒衰你雅波和样液到各自的微孔，每个标准波利详液应使刀新的吸头加人l.腹泻性贝类毒系酶标让物至母个微孔还速充分混台：222（避光处孵台:in：将微孔架倒管在吸水纸「拍打数欲·以保证完个除么微孔中的液体，个微孔汁人2洗液冲洗后扣干，再重复以上洗极梁作1次：剂人50n1发色剂牟每个微引中.轻拍混列，22（~2（黑暗避光处孵6min加人μ反成终山液至母个微孔中迅速混约片：在min内以牢气为率白调零测量关记录每个孔溶液15nm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
让微暖泻性贝类毒索标准液和液的平沟吸光度伯+安式（1)分别求得行个暖泻性贝类毒素标准液和空口液的门分比吸光妄值：
式中;
A——π分比吸光度值：
S腹泻性贝类系标准液或样液的平均吸光度恺：S,——的喂泻性贝类毒素录准液的乎均吸光度伯：5.6.2绘制标准由线
以百分比设光度侦（觉术级）为纵坐标：以腹泻性贝类毒索浓度（k）（对数级）为横坐标绘制出腹泻性贝类毒素标准液而分比吸光度估与腹泻烂贝类毒索浓度的标准出线，砾次试验均成重新绘制标催曲线。
5.6.3结果的计算
在5，5.2绘制的标准山线工样液门分比吸光度值所对应的腹泻性贝类寺素浓度即为试样中腹泻3
性贝类毒素含量（u/k）：
5.6.4结果的表述
当测定值小于10 8/k.刚授牛胶泻性贝类博素含小于 10 ng/kg：当测定侦大十等于10ng/k时：则报告实际测定数估6
测定低限
测定低限
本方法的测定低限为lcug/kg
6.2回收率
7健康和安全
S>/T1996—2007
f1何腹泻性贝类毒素含虽值大于16/16)~~20/16)的样品即敲认为是勺害的.对人类食用不安伞。
标准液含有腹泻贝类毒索·成特别小心·避接独反应终土波为1 mo/.硫酸.避免接触皮肤。SN/T1996—2007
Foreword
This Standard was propased hy and is under the charge af the National Regulatory Commission forCertificationand Accreditation.This Standard was drafted by Lioaning Entry-Exit Inspection and uarantine Buireau of the People'sRepublic of China and Dandong Entry-Exit Inspection and Quarantine Bureall.This Standard was rmnainly drarted by Yu Bing.Cao Jijuian.Gao Shiguang: Ma Lidan: Jin Dongquan.XieYan. Li Li and Sun Jie
This Standard was promulgated far the first timeS>/T 1996—2007
Determinationof diarrheticshellfishpoisoninshellfish-ELISAmethod
1Scope
This standard specifies the methods of sampling. sample preparation and ELisA for the determina-tion of diarrhetic shellfish poison.This standard is applicable to the determination of diarrlhetic shellfish poison in molluise meat: shellligament and other edible parts of bivalve for import and export. Positive results must be confirmedby orther methods.
2 Normative references
The following standards contain provisians, which are used in this text for reference. constituteprovisions of this Standard. For datec references, subsequent amendments (excluding corrections )or revisions of any of these publications shall not apply to this Standard. All parties subject to agree- ments based on this Standard are encouraged to investigate the possibility of applying the most re-cent editions of the standards indicated below, For undated references the latest editions of the pub-licationsrererredtoareapplicableSN o294 Method for the deternination of diarrhetic shellfish poisori in shellfish for import and export3 Terms,cefinitions and abbreviation3.1Terms and cefinition
The following terms and definitions are applicable to this Standard.3. 1.1 Diarrhetic shellish poison,DsPTypical cherrical constitutions are those of okadaic acid and it's rarrificatioris: a general term of tox-ic suhstances in halobios such as shellfish. which will consequently cause diarrhea in human heingswho eat the shellfish.
$N/T 1996—2007
3.2 abbreviation
Diarrhetic shellfish poinson.Dsp3.2.2
EnzymeLinked ImmunosorbentAssay.ELisA.Sampling andsamplepreparation4
Irriplementation is subject to sampling and sarriple preparation standard methods in sN o294Determination method
5.1Principle
This determination method is based on competing ELiSA reaction.: Free diarrhetic shellfish poisoncompetes with erizyme-labeled diarrhetic shellfish poison substance for diarrhetic shellfish poison an-tibody. The Lin-linked enzyme-labeled substance is removed in the process a[ washing. The enzymestroma anc chromogenic reagent are added into the well and incubatec. The linked enzyme-labeledsubstance then converts colorless chromogenic reagerit irito blue product. which will turn to yellowalter adding reactian stap salutian. Measure the absorptian value of solution in wells in 45onm wave-length and calculate the concentration of the diarrhetic shellfish poison in sample accorcing to thedrawri calibration curye.
Reagents and materials
All chemical reagents should be analytical reagents unless otherwise specified. Water should be di,distilled.
90% Methyl alcohol solution
Semilagarithmic peper.
DSP standard substarice.
Enzyme-labeled substance.
Diarrhetic shellfish poison antibody coated micropore plate5.2.6
Sustrate (urea peroxide): chrormogenic reagent (tetramethylbenzidine).5.2.7
Reaction stop salutian:1 mol/LH,s45.2.8
S>/T 1996—2007
Commercial kit that satisfies the requirements of determination limit and recovery in thisStandard is also applicable.5.3
Instrliments and equipments5.3.1
Enzyme-linked analyzer.
Homogjenizer,
Centrifuger:4'℃ 3 oa0g
5. 3, 4 Micro applicator: 50 μL5.3.5
Multi-channelmicroapplicator：50uL，10o r.L.5.4
Extraction and purification of sampleWeigh 10. d g sample (imprecision: 0. 1 g) . then add 5 times weight of 90% methyl alcohol solution.Homogenize the mixture [or 1 min~2 min. Centriluge at 4'C : 3 a00 g for 1a min. then transler thesupernatant into a new vial and dilute with didistilled water in ratio of 1 : 2 (1 - 1). Remove 50 μL ofdilution for ELiSA according to 5. 5. By now the extension rate is 10. For high-concentration samplewhose determination result exceeds the range of standard curve: further dilution should be donewith 90 % rriethyl alcohol solution uritil the coriceritration is within the rarige.5. 5Determination of ELISA
Insert adequate ELlsA strips into the micropore frame (double tests for standard and sample solu-tions respectively) and record their positions. Add 2 to 5 concentrations of standard solutions aswell as sample solutions of diarrhetic shellfish poison into different micropore (50 yiL each) usingnew tips, Then add 5D :L enzyme-laheled diarrhetic shellfish poison substance inta every micropareand mix sufliciently and quickly.Incubate the mixtures in dark at 22c~25 for 10 min. Bottom upthe frame on a piece of absorbent paper and flap it for several times so as to absolutely remove theliquid in the micropores. Wash the micropores with 250 rL washing solution each and flap the frameuntil nn liquid remained in the micropores. Repeat the nhove washing step for 4 times. Add 50 r.Lchromogenic reagent into every rricropore arid mix well by light flaps. Incubate the mixture in darkat 22'c ~25℃ for 6 min, Add 50 μL reaction stop solution into every micropore and mix well rapid-ly. Measure in 45b nm wavelength and record the absorption value of solution in every micropore af-ter setting the value of the empty micropore to zero for 1d min.5. 6Calcilation and Expression of resuilts5.6.1Calculation of the absorption percentageCalculate the average absorption vallies of diarrhetic shellfish poison standards and samples, work7
$N/T 1996—2007
out the percent absorption value af each standard and sample according to Formula (1):A
A percent absorptian value;
Saverage absorption value of diarrhetic shellfish poison staridard or sample:S.: average ahsorptian value af D μg/ L diarrhetic shellfish poison standard.5. 6. 2 Plotting standard curve1
By settirig the percent absorptiori values and the logarithrri concentration values of the diarrheticshellfish paison standardst g/kg) : plot the standard curve for percent absarption value and concen+tration of diarrhetic shellfish poison. Plotting should be done for every experiment.5. 6, 3 Calculation of resultThe diarrhetic shellfish poison concentration( μg/kg)of sarmple can be calculated according to its percent absorption value and the standard curve precised in 5. 6. 2.5. 6. 4 Expression of resuiltWhen the test result is 1o ry/kg.the concentration of diarrhetic shellfish poison should be repor.ted as 10 μg/kg.
When the test result is 1o μg/kg.the concentration of diarrhetic shellfish poison should be repor-ted according to thie exact value.Determinationlimitandrecovery6
Determination limit
The determination limit or this method is 1D rg/kg6.2 Recovery
The recovery of this method is 85% ~90%.7 Health and safety
Any sample whose concentration of diarrhetic shellfish poison above 16 μg/100 g~20 μg/100 g isconsidered to be harmful and Linsafe for human consumption.co
S>/T 19962007
Treat the standard solutions carefully and avoid contact since they contains diarrhetic shellfisl poi-son.
Avoidskincontactwiththereactianstopsolution.whichis1mol/LH,sO,SN/T 1996-2007
中华人民共和国业人境检验检收行业标准
贝类中腹泻性贝类毒素检验方法酶联免疫吸附法
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