1、叶己中华人民共和国出入境检验检疫行业标准SN /T 1119-2002 进口动物源性饲料中牛羊源性成分检测方法PCR方法Identification of bovine , sheep and goat derived materials in import animal derived feedstuff 一PCRmethod 2002 - 05 -20发布2002 -1个01实施中华人民共和国国家质量监督检验检痊总局发布SN /T 1119-2002 目次前言.m 引言.N 1 范围2 术语、定义和缩略语.3 抽样和制样.24 测定方法.3 5 结果及判断.4 6 废弃物处理和防止污染的措
2、施4附录A(规范性附录)确证试验-PCR扩增产物测序.5 附录B(规范性附录)检测过程中防止交叉污染的措施.6 参考文献.7 SN/T 1119-2002 前言本标准的附录A、附录B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。本标准起草单位:中国进出口商品检验技术研究所、中华人民共和国深圳出入境检验检疫局、中华人民共和国辽宁出入境检验检疫局、中华人民共和国珠海出入境检验检疫局。本标准主要起草人:徐宝梁、杨宝华、王静、曹际娟、薄清如。本标准系首次发布的出入境检验检疫行业标准。E SN/T 1119-2002 sl 牛海绵状脑病、痒病均可通过食用病畜组织传播，所以含牛羊源性成分的动
3、物源性饲料的使用，一直被认为是牛海绵状脑病、痒病得以传播的主要途径。禁止疫区含牛羊源性成分的动物源性饲料的生产、流通和使用，也就成为防止牛海绵状脑病、痒病感染、流行的主要手段。根据牛羊遗传物质的特异性，应用PCR方法，可以检测动物源性饲料中牛羊源性成分。本方法的测定低限为0.125%。N 1 范围进口动物源性饲料中牛羊源性成分检测方法PCR方法SN /T 1119-2002 本标准规定了进口动物源性饲料中牛羊源性成分检测的抽样、制样、PCR方法、限制性内切酶酶切反应方法。本标准适用于动物源性饲料中牛羊源性成分的定性检测。2 术语、定义和缩略语2.1 2.2 2.3 2.4 下列术语、定义和缩略
4、语适用于本标准。动物源性饲料animal derived feedstuff 含有动物成分的饲料。牛源性成分bovine derived material 牛特异性DNA片段。羊源性成分sheep and goat derived material 羊特异性DNA片段。聚合酶链式反应polymerase chain reaction 聚合酶链式反应，简称PCR。使用两段(20个24个核昔酸)寡核昔酸作为反应的引物，这两段寡核昔酸引物的序列应不发生互补作用。但它们可和称为模板的待测DNA两条链上在一定的位点分别发生互补。反应液由包括含有镜离子的反应缓冲液、4种单核昔酸(dNTP)、模板DNA及引
5、物。在DNA聚合酶催化下，通过温度的变化(DNA变性、退火及延伸)而合成两个互补位点之间的DNA片断。这样的反应反复进行，使第一个循环产生的DNA片断得以扩增。经25个30个循环，扩增倍数达1060、2.5 缩畸语PCR: polymerase chain reaction，简称PCR。DNA :deoxyribonucleic acid，脱氧核糖核酸。dNTP :deoxyribonucleoside triphosphate，脱氧核昔酸三磷酸。dATP :deoxyadenosine triphosphate，脱氧腺昔三磷酸。dCTP :deoxycytidine triphosphate
6、，脱氧胞昔三磷酸。dGTP :deoxyguanosine triphosphate，脱氧鸟昔三磷酸。dTTP :deoxythymidine triphosphate，脱氧胸昔三磷酸。dUTP :deoxyuridine triphosphate，脱氧尿昔三磷酸。UDG: uracil DNA glycosylase，尿暗院DNA-糖基酶。bp : base pair，碱基对。1 SN/T 1119-2002 BSA: bovine serum albumin，牛血清白蛋白。EDT A : ethylene diaminetetraacetic acid，乙二胶四乙酸。T aq : T he
7、rmus aquaticu，水生栖热菌。Tris : tris Chydroxymethyl) aminomethane，三(是甲基)氨基甲皖。TE: Tris-Cl、EDTA缓冲液。GuSCN :guanidinium isothiocyanate，异硫氨酸肌。Triton X-100:t-oct)句henoxypolyethoxyethanol.辛基苯氧基聚乙氧乙醇。3 抽样和制样3- 1 检验批以不超过200t为一检验批，简称批。同一检验批的商品应具有相同的特征，如包装、标记、产地、规格、等级。3. 2 抽样数量3. 2. 1 袋装饲料按式(1)计算抽样袋数:=1万式中:N一一全批袋数;
8、a 抽样袋数。注:a值取整数，小数部分向前进位为整数。3.2.2 散装饲料( 1 ) 根据散装单位的大小和类型，一般可从上、中、下三层的中心及四角五点抽样;或分层随机采样，取样点不少于15处。3.3 抽样工具3. 3. 1 金属单管抽样器:全长65cm75 cm，槽口长50cm55 cm，口宽1cml. 8 cm，头尖形，最大外径约2.5cm，或其他等效抽样器。3. 3. 2 取样铲。3.3.3 分样器。3. 3- 4 样品袋z可密封。3. 4 抽样方法3.4.1 袋装饲料将杆槽I臼下，从每袋一角依斜对角方向插入袋内，然后将杆槽旋转向上，抽出抨样器。每袋取样量不少于500g。每批所抽取的样品总
9、量不少于4kgo 3.4.2 散装饲料分层定点采样，每点取样量不少于500g。每批所抽取的样品总量不少于4kg。3. 4. 3 实验室样晶制备合并所取样品，充分混匀，用分样器或按四分法缩样，至样品重约1000 g。装入清洁容器内，加封后，标明标记，及时送交实验室。3.5 试样制备从所取实验室样品中取出有代表性样品约500g，经粉碎机粉碎，过20目筛，混匀，均分成三份。分别装入清洁容器内，加封后，标明标记。2 SN /T 1119-2002 3.6 试样保存试样于4C下避光保存。4 测定方法4.1 方法提要利用裂解液破碎细胞，二氯甲烧抽提蛋白质，异丙醇沉淀得到DNA;以提取的DNA为模板进行PC
10、R扩增，琼脂糖凝胶电泳检测PCR扩增产物;应用限制性内切酶酶切反应进行确证。4.2 试剂和材料除另有规定外，试剂为分析纯或生化试剂，水为灭菌双蒸水。4.2.1号|物4.2.1.1 牛源性成分检测用引物(对)序列为z5 -GCCATATACTCTCCTTGGTGACA-3 5 -GTAGGCTTGGGAATAGTACGA-3 4.2.1.2 羊源性成分检测用引物(对)序列为:5 -T A TT AGGCCTCCCCCTTG TT -3 5 -CCCTGCTCAT AAGGGAAT AGCC-3 4. 2. 2 Taq DNA聚合酶。4.2.3 限制性内切酶z峙nn , sau3A 1 。4.2.
11、4 dNTP :dATP、dTTP、dCTP、dGTP。4.2.5 琼脂糖:电泳纯。4.2.6 澳化乙键。4.2.7 三氯甲:皖。4.2.8 异丙晖。4.2.9 70%乙醇。4.2.10 分子量标记:50bp300 bp。4.2.11 裂解液:5 mol/L GuSCN , 0. 05 mol/L Tris-盐酸(pH6.的，0.02mol/L EDTA(pH8. 0) ,1.3% Triton X-100。4.2.12 TE缓冲液:10 mmol/L Tris-盐酸(pH8.0) , 1 mmol/L EDTA(pH8. 0)。4.2.13 10XPCR缓冲液:100mmol/L氯化饵，16
12、0mmol/L硫酸钱，20mmol/L硫酸镜，200mmol/L Tris-盐酸(pH8.肘，1%Triton X-100 , 1 mg/mIBSA。4.2.14 电泳缓冲液:Tris 54 g，棚酸27.5g , O. 5 mol/L EDTA(pH8. 0)20 mL，加蒸馆水至1000mL; 使用时10倍稀释。4.2.15 加样缓冲液:0.25%澳酣蓝，40%蔚糖。4.2.16 酶切缓冲液:10 mmol/L Tris-HCl (pH7. 5) , 10 mmol/L氯化镖，50mmol/L氯化锅，0.1 mg/mL BSA。4.3 仪器和设备4.3.1 粉碎机。4.3.2 离心机。4.
13、3.3 DNA热循环仪。4.3.4 电泳仪。4.3.5 pH 计。4.3.6 移液器:10L、20L、100L、1000Lo 4. 3. 7 紫外检测仪。3 SN/T 1119-2002 4.3.8 恒温水浴锅。4.4 步骤4.4.1 模板DNA提取称取50mg试料于1.5 mL离心管中，加人200LTE，混匀;加入400/.LL裂解液，加400L三氯甲烧，混匀;10 000 r/min离心5min，取上清液;加0.8倍体积异丙醇，沉淀;10 000 r/min离心5min , 弃上清破;70%乙醇洗涤一次，晾干才日入50LTE，珞解沉淀。也可用等效DNA提取试剂盒提取模板DNA。4.4.2
14、PCR扩增反应体系体积为50L:10XPCR缓冲液5L、dNTP(5 mmol!L) 1L， 31物对(各5mol!L)2L、TaqDNA聚合酶(5U/L)0.5L、模板DNA(O.lgl.0吨)10L，Jc31. 5L。反应条件:94C预变性1min3 mino 94C变性30s60 s.56C58C退火30s60 s , 72C延伸30s60s，30个循环。72C延伸5mino 4C保存。检测过程中分别设阳性对照、阴性对照和空白对照。用牛源性饲料或羊源性饲料作阳性对照，用非牛源性饲料或非羊源性饲料作阴性对照，空白对照除不加模板外，测定步骤相同。4.4.3 PCR扩增产物电泳检副取1.5g琼
15、脂糖，于100mL电泳缓冲液中加热，充分溶化，加入澳化乙键至终浓度为1g/mL.制胶。在电泳槽中加入电泳缓冲液，使液面刚刚没过凝胶。将5L8LPCR扩增产物分别和适量加样缓冲液混合，点样。9V/cm恒压，电泳10min20 min。紫外检测仪下观察电泳结果并记录。4.4.4 确证试验4.4.4.1 限制性内切酶酶切反应PCR扩增产物电泳检测结果阳性，进行限制性内切酶酶切反应。Dpn 1I反应体系(50L):D户n1I酶(5U/L)2L，酶切缓冲液5L，PCR扩增产物20L.水23L Sau3 A I反应体系(50L):Sau3 A I酶(10U /，uL)lL，酶切缓冲液5L，PCR扩增产物2
16、0L.水24L。充分泪匀反应液.3TC温浴1h，65C水洛5min终止酶切反应。取2g3 g琼脂糖，其余同4.4.3，进行限制性内切酶酶切产物电泳检测。4.4.4.2 PCR扩增产物测序必要时，进行PCR扩增产物测序，具体步骤见附录A。5 结果及判断5.1 PCR扩增产物电泳检测结果牛源性成分的PCR扩增产物为271bp，羊源性成分的PCR扩增产物为294bp。5.2 限制性内切酶酶切产物电泳检测结果牛源性成分的PCR扩增产物乓户n1I限制性酶切产物为57bp、214bp 0羊源性成分的PCR扩增产物Sau3A I限制性酶切产物，绵羊为91bp、203bp，山羊为92bp、202bp 0 5.
17、3 结果表述PCR扩增产物电泳检测结果阴性，未检出牛或羊源性成分。PCR扩增产物电泳检测结果阳性，限制性内切酶酶切产物片段大小正确，检出牛或羊源性成分。6 废弃物处理和防止污染的措施4 检测过程中的废弃物，收集后在焚烧炉中焚烧处理。检测过程中防止交叉污染的措施见附录B。A.1 试剂和仪器A. 1- 1 PCR扩增产物纯化试剂盒。A.1.2 DNA测序试剂。A.1.3 95%乙醇。A.1.4 甲酷肢。A.1.5 DNA序列分析仪。A.2 步骤A.2.1 PCR扩增产物的纯化附录A(规范性附录)确证试验-PCR扩增产物测序按PCR扩增产物纯化试剂盒要求纯化，或直接测序扩增。A. 2. 2 测序扩增
18、反应SN/T 1119-2002 反应体系(20L):8LDNA测序试剂，200ng500 ng PCR纯化产物，3.2pmol引物，水补足至20L;PCR扩增程序:960C10 s , 50C 5 s , 60C 4 min，25个循环，扩增产物4C保存。A.2.3 测序扩增产物的纯化扩增管中加入16L水、64L95%乙醇，稍混匀，室温放置15min , 12 000 r/min离心20min，去上清，加入250L70%乙醇，短暂混匀，12000r/min离心10min，去上清，室温干燥。A.2.4 测序纯化产物管中加入170L甲酷胶榕液，95 C , 5 min，迅速转移至冰上，2mino
19、分装样品于测序仪的加样槽中，自动测序。A.3 PCR扩增产物测序结果A. 3. 1 牛源性成分的PCR扩增产物序列gtaggcttgggaa tagtacga taagggctacgagagggagacctaaaa ttacaggggtaa taaaagaggtaaa taaa ttttcgttca t ttt g tttctcaaggggtgt tt tgttttaa ta tttttgttggtgtcagttctgga ttgtga taaaggttgtgttttgaaacttttagt tgaaaga tga taa aaagggtcaagaa ta ttga taaga tca ttg
20、tcagtca tgttgacgtgtc tagttgcggca t gtcaccaaggagagta ta tggc A.3.2 羊源性成分的PCR扩增产物序列A. 3. 2. 1 绵羊ccctgctca taagggaa tagcca tgcc taggttta ttga tagtt gtgtagttggtgtaaa tgagtggggtaggaggcctagtaggtttgtaga t ccaa taaa taaaa ttagggaca ttagta ttaa tagctca tgtctgtcct ttggtgtta tgaa tgctca tta tttgttttga tactaa t
21、tgaagta ttc act gttggagggaga tgaggcaggttgttgact agtcggcttga tgtgggaaa taa taggctagggaa taaaacaa tgagggtaacaaggg ggaggcctaa ta A. 3. 2. 2 山羊ccctgctca taagggaa tagccca tgcctaga ttta ttga tagttgtgtagttggtgtaaa tgagtggggtagaaggcctaa taggtttgtaga tccaa taaa tagga ttagggaca ttaa ta ttaa tgttca tgtttgtcctt
22、tggtgtta tgaa tactta tta tttgttttga tatgagttgaagtgccc attgttggagagagacgaggcggttgttaattagtcggtttgatgagggaaatagtaagctaggaaataaaataataagggtaacaagggg gaggcctaata 5 SN /T 1119-2002 附录B(规范性附录)检测过程中防止交叉污染的措施B.1 抽样和制样过程抽样和制样工具，必须清洗干净，121C、15min20 min高压，一套清洁工具限于一个样品使用。存放样品的容器应该经过清洗、高压，或为一次性灭菌容器。B.2 检测过程B.2.1
23、 PCR实验室应分为样品制备区、前PCR区、PCR区、后PCR区。将模板提取、PCR反应液配制、PCR循环扩增及PCR产物的鉴定等步骤分区或分室进行。实验室的运作应从净区到脏区单方向进行。B. 2. 2 实验过程中，必须穿实验服和戴手套，手套要经常更换。各区要有专用实验服，经常清洗。B. 2. 3 各区所有的试剂、器材(尤其是移液器)、仪器都应专用，不得带出该区。B. 2.4 所有溶液、水、耗材和器具要121C、15min20 min高压，避免核酸和(或)核酸酶污染。每种溶液必须使用高质量的成分和新蒸馆的双蒸水。在20C25C贮存的试剂中，可加入0.025%的叠氮铀。所有试剂应该以大体积配制，
24、然后分装成仅够一次使用的量进行贮存。B. 2. 5 装有DNA模板或引物的离心管打开之前，要简短离心，离心管不能用力崩开，以免产生气溶胶。B. 2. 6 前PCR区中，最好能在PCR操作箱中加入PCR反应各组分。B. 2. 7 实验前后，实验室用紫外线消毒以破坏残留的DNA。B. 2. 8 可使用UDG和dUTP系统控制污染。B. 2. 9 应遵循PCR操作的其他要求。6 SN /T 1119-2002 参考文献lJ Tartaglia ,M. ,Saulle ,E. ,et al. (1 998). Detection of Bovine Mitochondrial DNA in Rumin
25、ant Feeds: A Molecular Approach to Test for the Presence of Bovine-Drived Materials. ournal of Food Protection , 61: 513-518. 2J Wang ,R.G. .My巳rs,M. ,et al. (2000). A Rapid Method for PCR Detection of Bovine Meterials in Animal Feedstuffs. Molecular and Probes. 14: 1-5. 3J C. W.迪芬巴赫，G.S.德维克斯勒(著).黄培
26、堂，俞炜源等(译).PCR技术实验指南.科学出版社，1998，140.7 SN/T 1119一2002Contents Foreword 9 Introduction 10 1 Scope 11 2 Technical terms , definitions , abbreviations 11 3 Sampling and sample preparation 12 4 Method of detection 13 5 Results and final conclusions 16 6 Treatment of waste and measures to prevent cross con
27、tamination in identification 16 Appendix A( Normative) Verification experiment-Sequencing of PCR product 17 Appendix B( Normative) Measures to prevent cross contamination in identification 19 Reference 20 8 SN/T 1119一2002Foreword The annex A and annex B in the standard were normative annexes. This s
28、tandard was proposed by National Regulatory Commission for Certification and Accredita tion. This standard is under the charge of National Regulatory Commission for Certification and Accredi tation. China Import and Export Commodity Inspection Technology Institute, Shenzhen Entry - Exit Inspec tion
29、and Ouarantine Bureau of The People 5 Republic of China , Liaoning Entry-Exit Inspection and Ouarantine Bureau of The People 5 Republic of China , Zhuhai Entry - Exit Inspection and Ouaran tine Bureau of The People 5 Republic of China drafted this standard. This standard was mainly drafted by Xu Bao
30、liang , Yang Baohua , Wang Jing , Cao Jijuan , Bo Oin gru. This standard is a professional standard promulgated of Entry-Exit inspection and quarantine for the first time. Note: The English version , a translation from the Chinese text , is for guidance solely. 9 SN/T 1119-2002 Introduction Bovine s
31、pongiform encephalopath(BSE) and scrapie both can be spread by eating of infected tissues. The use of animal-derived feedstuff that contains bovine , sheep and goat-derived material has been regarded as the main media to spread BSE and scrapie. To forbid the production , circula tion and use of anim
32、al - derived feedstuff containing bovine , sheep and goat - derived materials from the epidemic region is an important measure for control of infection and epidemic of BSE and scraple. The bovine , sheep and goat - derived material in animal - derived feedstuff can be identified by PCR method on the
33、 basis of difference of genetic material of bovi ne , sheep and goat. The lowest limit of this method is 0.125%. 10 SN/T 1119一2002Identification of bovine , sheep and goat material in import animal - derived feedstuff-PCR method 1 Scope This standard specifies the methods of sampling , sample prepar
34、ation , PCR and restriction endonu clease analysis for identification of bovine, sheep and goat material in impo内animalderivedfeed甸stu忏.This standard is applicable to the identification of bovine , sheep and goat material in import animal - derived feedstuff. 2 Technical terms, definitions, abbrevia
35、tions The technical terms , definitions, abbreviations below are applicable to the standard. 2.1 Animal- derived feedstuff The feedstuff containing animal derived materials. 2.2 Bovine - derived material Bovine characteristic DNA fragments. 2.3 Sheep and goat - derived material Sheep and goat charac
36、teristic DNA fragment. 2.4 Polymerase chain reaction The Polymerase chain reaction (PCR) is a technique which is used to amplify the number of copies of a specific region of DNA. Two oligonucleotides compose by 20 - 24 nucleotides are used as primers. The sequences of two primers complement with spe
37、cial sequence in one of the strand of DNA which so - called template DNA but not complement each other. Reaction mixture consists by reaction buffer containing Mg2+ , four mononucleotides (dNTP) , template DNA and primer. Under the catalysis of DNA polymerase, the DNA fragments between two complemen
38、tary sties are syn thesized during denaturation , annealing and elongation of DNA reaction. The new DNA fragments become new templates in the next cycle and are propagated later on. The copy number of DNA fragment can reach to 106 after 25 - 30 cycles. 11 SN/T 1119-2002 2.5 Abbreviations PCR: polyme
39、rase chain reaction. DN.A: deoxyribonucleic acid. dNTP: deoxyribonucleoside triphosphate. dATP: deoxyadenosine triphosphate. dCTP: deoxycytidine triphosphate. dGTP: deoxyguanosine triphosphate. dTTP: deoxythymidine triphosphate. dUTP: deoxyuridine triphosphate. UDG: uracil DNA glycosylase. bp: base
40、pair. BSA: bovine serum albumin. EDTA: ethylene diaminetetraacetic acid. Taq: Thermus aquaticu. Tris: tris (hydroxmethyl) aminomethane. TE: Tris-CI , EDTA buffer. Triton X-1 00: t-octylphenoxypolyethoxyethanol. GuSCN: guanidinium isothiocyanate. 3 Sampling and sample preparation 3. 1 Inspection lot
41、The quantity of an inspection lot should not be more than 200 t , hereinafter referred to as lot . The characteristics of the cargo within the same inspection lot, such as packing , mark, origin , speci fication , grade etc. , should be identical. 3.2 Amount of sampling. 3.2. 1 Feedstuff in package
42、The number of package to be taken is calculated by following formula: where N 一一一totalnumber of package in a lot a一-numberof package to be taken a = -1而.( 1 ) Note: if value a is with decimal , round off the decimal part , which is added as unity to the intergal part of a. 3.2.2 Feedstuff in bulk Ac
43、cording to the size and type of the bulk unit, the sample can be generally taken from the center and four corners of upper, middle and low layers. Or taken from different layers at random , the number of the spots for sampling should not be less than 15. 3.3 Sampling tool 12 SN/T 1119-2002 3.3.1 Sha
44、rp - pointed metal tube with one notch: length: 65 cm - 75 cm; length of notch: 50 cm -55 cm;width of notch:1 cm - 1.8 cm;maximum diameter:2.5 cm approximately.Or equivalent. 3.3.2 Shovel. 3.3.3 Sample divider. 3.3.4 Samples bags: can be sealed. 3.4 Sampling procedure 3.4. 1 Feedstuff in package Sam
45、ple is taken from each package by insert the tube from the comer of the package to the direc tion of the diagonal corner with the notch downward. Upturn the tube till the notch upward and pull the tube out to finish the sampling. The total weight of sample of each package should be not less than 500 g. The total weight of sample of each lot should be not less than 4 kg. 3.4.2 Feedstuff in bulk As the same as 3.2.2, the weight of samples at each spot should be not less than 500 g. The total weight of sample of each lot should be not less than 4 kg. 3.4.3 Preparation of lab samp
