【行业标准】 动物源性食品中甲砜霉素和氟甲砜霉素药物残留检测方法 微生物抑制法

中华人民共和国出入境检验检疫行业标准SN/T2423—2010
动物源性食品中甲霉素和
氟甲砜霉素药物残留检测方法
微生物抑制法
Determination of thiamphenicol and florfenicol residuesin animal derived foodMIA method2010-01-10发布
中华人民共和国
国家质量监督检验检疫总局
合绿伴网
2010-07-16实施
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。SN/T2423—2010
本标准起草单位：中华人民共和国广东出入境检验检疫局、中华人民共和国湛江出人境检验检疫局。本标准主要起草人：王志强、李志勇、许龙岩、凌莉、陈宗辉、陈春梅本标准是首次发布的出入境检验检疫行业标准I
http：//foodmate.net1范围
动物源性食品中甲霉素和
氟甲霉素药物残留检测方法
微生物抑制法
SN/T2423—2010
本标准规定了动物源性食品中甲砜霉素和氟甲砜霉素药物残留检测的微生物抑制法。本标准适用于肉类、鱼、虾和牛奶中甲砜霉素和氟甲砜霉素药物残留筛选检测。2规范性引用文件
下列文件中的条款通过本标准的引用而成为本标准的条款。凡是注日期的引用文件，其随后所有的修改单（不包括勘误的内容）或修订版均不适用于本标准，然而，鼓励根据本标准达成协议的各方研究是否可使用这些文件的最新版本。凡是不注日期的引用文件，其最新版本适用于本标准。GB/T6682分析实验室用水规格和试验方法3设备和材料
3.1设备
3.1.1电子天平：灵敏度为0.01g。3.1.2高速离心机。
3.1.3高速组织捣碎机。
涡旋振荡器。
恒温水浴锅。
游标卡尺：测量范围0mm200mm，精度0.02mm或抑制圈测量仪。灭菌平：直径90mm，底部平整的玻璃或一次性塑料灭菌平皿。圆形滤纸片：直径1omm，厚1.1mm。日本ToyoRoshiKaisha，Ltd.产品或相当者。50mL离心管。
可调移液器：10μ~100μ，100μ~1000μ3.1.11
恒温箱：37℃±1℃。
麦氏比浊计。
3.2菌种和培养基
3.2.1藤黄微球菌MicrococcusluteusATCC934l3.2.2纯化及传代培养基（TSA)：见附录A第A.1章。3.2.3检定用培养基：抗生素培养基1号（AMI)见附录A第A.2章。3.3试剂
本方法所用化学试剂为分析纯，试验用水为蒸馏水或去离子水，试验用水应该符合GB/T6682中一级水的规定，
3.3.1pH6.0磷酸盐缓冲液
pH6.0磷酸盐缓冲液：称取8.0g磷酸二氢钾（KH2PO4）及2.0g磷酸氢二钾（K2HPO4），用蒸馅水溶解并定容至1000mL，121℃高压灭菌15min。3.3.2pH6.0磷酸盐缓冲液-乙睛提取液pH6.0磷酸盐缓冲液和乙腈按2：3体积比制备。1
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3.3.3甲砜霉素标准储备液
称取一定量的甲砜霉素标准品，用甲醇溶解并定容至1000ug/mL置2℃～8℃冰箱保存，贮存期6个月。
3.3.4甲砜霉素标准工作液
吸取一定量的甲砜霉素标准储备液，用pH6.0磷酸盐缓冲液稀释成0.01ug/mL的标准工作液！应当日配制使用
3.3.5氟甲霉素标准储备液
称取一定量的氟甲霉素标准品，用甲醇溶解并定容至1000ug/mL.置2℃～8℃冰箱保存，贮存期6个月。
3.3.6氟甲霉素标准工作液
吸取一定量的氟甲霉素标准储备液，用pH6.0磷酸盐缓冲液稀释成0.02ug/mL的标准工作液，应当日配制使用。
4检验方法
4.1方法提要
将测试样品经缓冲液提取、温育和离心后，取上清液进行测定。利用抗生素对敏感菌的抑制作用来判别试样中是否含有甲砜霉素和氟甲砜霉素类抗生素。4.2试样制备和保存
4.2.1试样制备
肉、鱼和虾：从原始样品中取出部分有代表性样品，取250g可食部分放人高速组织捣碎机均质，充分混匀，装入清洁容器内，并标明标记。牛奶同批次直接从三个独立包装客取100mL混合均匀。装入清洁容器内，并标明标记
测试样品制备操作过程中应防止样品受到污染或发生残留物含量的变化。4.2.2试样保存
肉、鱼、虾：试样于一18℃以下保存，牛奶2℃～8℃保存4.3菌悬液制备
将藤黄微球菌ATCC9341菌种用普通营养肉汤培养基复活，接种血平板分离出单个菌落，挑取单个菌落接种纯化及传代培养基（TSA)斜面，37℃培养18h～24h。取斜面菌苔制成0.5麦氏单位菌悬液备用。
4.4检定用藤黄微球菌ATCC9341平板制备无菌吸取0.5mL0.5麦氏单位藤黄微球菌悬液注人灭菌平Ⅲ内，加人8mL溶化后冷却至50℃左右灭菌抗生素培养基1号（AMI)培养基.充分混勾后保持水平使其凝固，放置42℃温箱中烘干15min备用.所用检定平板应当天制备。4.5测定步骤
4.5.1样液提取
称取10.0g均质好的试样于50mL离心管中，加人20mLpH6.0磷酸盐缓冲液-乙睛提取液，用涡旋振荡器充分振荡3min，于65℃～70℃水浴保温30min后，3000r/min离心15min取上清液作为样液，牛奶样品直接取样。
4.5.2测定
取制备好的检定用平板各两个，在平板底部做好标记，把滤纸片适当间隔置于平板上，每个平板最多不超过4个.用镊子轻压使其固定，在每片滤纸上滴加100pL样液或标准工作液.静置30min后.置37℃培养18h后观察，用游标卡尺测量抑菌圈直径大小，每份样品做两个平板平行样试验。2
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4.6检测结果判定和报告
SN/T2423—2010
4.6.1检定平板上待测样品不产生抑菌圈或抑菌圈直径≤10mm，标准工作液的抑菌圈>14mm，报告未检出甲砜霉素、氟甲砜霉素。4.6.2检定平板上待测样品产生抑菌圈，抑菌圈直径≥12mm，标准工作液的抑菌圈>14mm，报告初筛检出甲砜霉素、氟甲砜霉素
4.6.3检定平板上待测样品产生抑菌圈：10mm<抑菌圈直径≤12mm，标准工作液的抑菌圈>14mm，判为可疑，必要时重新取样检测。第二次检测结果为阳性或可疑时，报告初筛检出甲矾霉素、氟甲霉素，为阴性时,报告未检出甲砜霉素、氟甲砜霉素。检测限
5.1肉类、鱼和虾样品：
甲矾霉素50μg/kg
氟甲砜霉素100ug/kg
5.2牛奶样品：
甲矾霉素20μg/kg；
氟甲砜霉素50ug/kg
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SN/T2423—2010
A.1纯化及传代细菌培养基（TSA）A.1.1
胰蛋白陈
植物蛋白陈
氯化钠
蒸馏水
pH7.3±0.2
A.1.2制法
附录A
（规范性附录）
1000mL
将各成分加热溶解，分装试管，121℃高压灭菌15min，制成斜面。注：制成半固体时，琼脂量为0.5%。抗生素培养基1号（AMI）
A.2.1成分
牛肉膏
蛋白陈
磷酸氢二钾
蒸馏水
pH6.6±0.2
A.2.2制法
1000mL
将各成分加热溶解，分装每瓶100mL，121℃高压灭菌15min。http：//foodmate.netForeword
AnnexAof thisstandard is normativeannexsSN/T2423—2010
This standard was proposed by and is under the charge of National Regulatory Commission for Certi-ficationandAccreditation
This standard was drafted by Guangdong Entry-Exit Inspection and Quarantine Bureau, ZhanjiangEntry-Exit InspectionandQuarantineBureauof thePeople'sRepublicofChinaThis main drafters of this standard are Wang zhiqiang,Li zhiyong,Xu longyan,Ling li,Chen zonghui,Chen chunmei.
This standard is a professional standard for Entry-Exit Inspection and Quarantine promulgated for thefirsttime.
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SN/T 2423—2010
Determination of thiamphenicolandflorfenicol residuesinanimalderivedfoodMlAmethod1
The standard specifies the microbial inhibition assay method for detection of antibiotic residues inanimalderivedfoods
This standard is applicable to the screening inspection of antibiotic residues in meat.fish,shrimp andmilk.
2Normative reference
The following normative documents contain provisions which,through reference in this text,consti-tute provisions of this standard. For dated reference,subsequent amendments to (excluding editingcorrections).orrevisionofanyofthesepublicationsdonotapply.However,parties to agreements based on this standard are encouraged to investigate the possibilityof applying the most recent edition of the standards indicated below.For undated references,thelatesteditionofthenormativedocumentreferredtoapplies.GB/T 6682Water for analytical laboratory use-Specification and test methods3
Equipments and materials
Equipments
Electronicscalewithasensitivityofo.o1g.3.1.1
Centrifuge
High-speed tissue blender.
Vortexmixer
Waterbath
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SN/T2423—2010
3.1.6Vernier caliper,0 mm~200 mm range,with a precision of 0. 02 mm; or inhibiting circlemeter.
Sterilized Petri dishes,90 mm diameter.glasses or plastic with flat and smooth bottom.3.1.7
3.1.8Filter paper disk,10 mm diameter,1 mm thickness.Product of Toyo Roshi Kaisha Ltd,JapanorEquipment
Centrifugaltube:5omL.
Finnpepette:10μL~100μL,100μL~1000μLIncubatorsetat37℃±1.theinterlayersshouldbehorizontall3.1.12
Nephelometer
Reagents and supplies
3.2.1MicrococcusluteusATcc93413.2.2Media for preservation and transportation of bacteria(TSA):RefertoannexA.1.3.2.3Mediaforbioassay：RefertoannexA.2.3.3
Reagents
All chemical reagents used in this method are of analytically pure. Test Water should be distilledwaterordeionizedwaterinconformitywithGB/T66823.3.1pH6.0phosphatebuffer
Weighout8.0gpotassiumdihydrogenphosphate(KH2PO4)and2.0gdipotassiumhydrogenphos-phate (K2HPO4),dissolve in and bring the volume of the solution to 100 mL with distilled water.Autocalveat121℃for15min.
3.3.2pH6.0phosphatebuffersolution-acetonitrileMixedacetonitrile+phosphatebufferinaratioof3+23.3.3Thiamphenicol reference stock solutionWeigh out a certain quantity of thiamphenicol,dissolve in and bring the concentration to1 0oo μg/mL with methanol.The stock solution should be stored in refrigerator at 2℃ ~8 ℃ and7
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canbeusedfor6months.
3.3.4ThiamphenicolreferenceworkingsolutiorPipette a certain quantity of thiamphenicol stock solution,and dilute it to O.o1 μg/mL referenceworking solution with pH 6.O phosphate buffer.The working solution should be used in the same dayofpreparation.
3.3.5Florfenicol referencestocksolutionWeigh out a certain quantity of florfenicol,dissolve in and bring the concentration to 1 ooo μg/mLwith methanol. The stock solution should be stored in refrigerator at 2 C ~8 ℃ and can be used for6months.
3.3.6FlorfenicolreferenceworkingsolutionPipette a certain quantity of florfenicol stock solution,and dilute it to 0.02 μg/mL reference workingsolutionwithpH6.Ophosphatebuffer.Theworkingsolution shouldbeused inthesamedayof preparation.
Assaymethod
Brief introduction
Test sample is distilled with buffer solution,incubated and centrifuged. Take supernatant for test.Utilized the inhibiting effect of antibiotics on sensitive bacteria to determine if there are antibioticsinthetestsample.
Samplepreparation andstorage4.2.1Samplepreparation
Meat,fish and shrimp:Take a representative part from the original sample,place the edible portion(250 g) in high-speed tissueblenderto homogenize and blend the sample thoroughly.The same batchofmilk from three separate packages（100 mL)aremixed evenly.Place the mixture in a cleancontainer and mark the container.When preparing samples,measures should be taken to prevent sample from being contaminated orcontentof residuebeingchanged.8免费标准下载网bzxz
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4.2.2Samplepreservation
Meat,fish,shrimp:storedat-18℃orbelow.milk:2℃~8℃4.3Preparingbacterial suspensionsSN/T2423—2010
Bacterialsuspensionof micrococcusluteus ATcc9341:Inoculate resurgent bacteriain bloodflatmedium, then take single colony to TSA slope medium for reproduction of bacteria. Incubate at 37 ℃for 18 h~24 h. Take the lawn to prepare the bacterial suspension (0.5 Mcfarland unit).4.4PreparingmicrococcusluteusATCC9341bioassayplateAdd 0.5 mL of bacterial suspension (0.5 Mcfarland unit) to plate,then add AMl. That has beendissolved,autoclaved and cooled to about 50 C,and mix them well. Keep the plate horizontal untiltheagarsolidified.ThepreparedplateShouldbeused inthesamedayofpreparation.4.5 Assay procedure
Extractingsampleliquid
Weigh out 10.0 g homogenized sample and put it in a 50 mL centrifuge tube. Add 20 mL of acetoni.trile-phosphatebuffersolution.Vibrateinvortexmixerfor3minto ensurethoroughmixing.Incubatein water bath at 65 ℃ ~70 ℃ for 30 min, centrifuge at 3 000 g r/min 15 min,and then pipette thesupernatant as sample liquid.Formilk,pipette directly4.5.2Performingthebioassay
Take each two of thekinds of plates.Mark the plates on thebottom surface.Placefilter paper disksonplates inasuitableseparationdistance(nomorethan4diskforoneplate).Pressthefilterpapergently to keep them in place with a tweezers.Drop 100 μL of sample liquid or reference workingsolution on eachfilterpaperdisks.Then incubation at37℃for18h.Performparallel tests.Foreachsample on two disks.
4.6 Determination and reporting assay result4. 6. 1In case of a sample does not produce an inhibiting circle on any plate or produces an inhibi-ting circle with a diameter smaller than 10 mm while the reference working solution larger than14mm.Theassay result isnegative.4.6.2In case of a sample produces an inhibiting circle with a diameter 12 mm or larger.\screeningassayresultpositive\should bereported.9
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In case of a sample produces an inhibiting circle,and the diameter is larger than 10 mm and4.6.3
smaller than12 mm.The assay result is suspicious.If necessary,re-perform theassay for determina-tion.
Limit of determination
Meatfish and shrimp:
Thiamphenicol:50μg/kg
—Florfenicol:100μg/kg
—Thiamphenicol:20μg/kg;
—Florfenicol:50μg/kg.
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