1、中华人民共和国出入境检验检疫行业标准SN/T 1635-2005 贝类中诺沃克病毒检测方法普通RT-PCR方法和实时荧光RT-PCR方法Detection of norovirus in sheIlfish一Conventional RT-PCR and real皿timeRT-PCR 2005皿08-18发布060608000096 2006-02-01实施中华人民共和国发布国家质量监督检验检菇总局SN/T 1635-2005 前本标准的附录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。本标准起草单位z中华人民共和国广西出人境检验检疫局、中华人民共和国上海出入境检验检疫局。本
2、标准主要起草人z刘牢义、潘良文、张舒亚、李晓虹、韦梅良、罗兆飞。本标准系首次发布的出入境检验检疫行业标准。I SN/T 1635-2005 贝类中诺沃克病毒检测方法普通RT-P方法和实时荧光RT-Pl方法1 范围本标准规定了贝类中诺沃克病毒普通R子PCR和实时荧光RT-PCR检扭u方法。本标准适用于贝类中诺沃克病毒的检测。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。凡是注目期的引用文件，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堕文件的最新版本。凡是不注目期的引用文件，其最新版本适用于本标准。SN/
3、T 1193 基因分析检测实验室技术要求3 术语和定义下列术语和定义适用于本标准。3. 1 实时荧光RT-PCRreal-time fluorescence RT-PCR 实时荧光RT-PCR方法是在常规RT-PCR的基础上，加入了一条特异性的荧光探针。该探针为一寡核昔酸，两端分别标记一个报告荧光基因和一个摔灭荧光基因。探针完整时，报告基面发射的荧光信号被摔灭基因吸收;PCR扩增时，Taq酶的5-3外切梅活性将探针酶切降解，使报告荧光基固和摔灭荧光基团分离，从而荧光监测系统可接收到荧光信号，即每扩增一条DNA链，就有一个荧光分子形成，实现了荧光信号的累积与PCR产物形成完全同步。3.2 Ct值
4、cyclethreshold 每个反应管内的荧光倍号达到设定的域值时所经历的循环数。4 方法提要用Tri-reagent或其他等效裂解液提取病毒RNA，并根据诺沃克病毒RNA3末端含有一个PolyCA)的结构，用含有PolyCdD的磁珠吸附诺沃克病毒RNA进行进一步纯化。利用普通R下PC卫和实时荧光RT-PCR进行检瓣，对可疑的PCR产物进行测序分析确证。5 试都所有实验用试剂均为分析纯;除特别说明外，实验用水为蒸锚水或去离子水。5. 1 诺沃克病毒阳性标本:由自家质量监督检验检疫总局指定单位提供。一80.C低撮冰箱保存。5.2 甘氨酸缓冲液z见附录A.1.l.5.3 PEG 8 000溶液z
5、见附录A.1. 20 5.4 裂解被:Tri-reagent或其他等效裂解液。5.5 PolyCdT)磁珠:Dynabeads甲oligoCdT)25或等效品。5.6 元RNase超纯水z见附录A.2. 30 1 SNjT 1635-2005 5.7 75%乙醇z见附录A.2. 40 5.8 异两百享:未开封的新品。5.9 1XRNA吸附缓冲踉z见附录A.2. 50 5.10 2XRNA吸附缓冲液z见附录A.2. 6 0 5. 11 洗液z见附录A.2. 7 0 5.12 RNase抑制剂。5. 13 逆转录酶AMVo5.14 DNA聚合酶。5.15 dNTPs:含dATP、dUTP, dCT
6、P ，dGTP各10mmol/L。5. 16 引物和探针z根据表1和表2的序列进行合成引物和探针，加元RNase超纯水配制成50mol/L储存液。5.17 DNA分子量标记:100 bp2 000 bpo 5.18 50XTAE援冲液z见附录A.1.305. 19 澳化乙链溶液(10gjL):见附录A.1. 40 5.20 含0.5g/mL澳化乙链的1.5%琼脂糖凝胶=见附录A.1. 5。5.21 10X加样缓冲液z见附录A.1. 6 0 表1普通RT-PCR检测的引物检测的病毒类群l 引物扩增片断大小/bpGI和GU检测的病毒类群GI GU 6 仪器与器材正义寻i物JV12:5人ATACCA
7、CT ATGATGCAGATT A-3 反义引物JV13，5人TCATCATCACCATAGAAAGAG-3 表2实时荧光RT-PCR检测的引物和探针引物和探针序列正义寻i物COG1F:5-CGYTGGATGCGNTTYCATGA-3 反义引物COG1R:5人CTTAGACGCCATCATCATTYAC-3 探针RINGl(a)-TP: 5人FAM-AGATYGCGATCYCCTGTCCA-T AMRA叩3探针R卧G1(b)-TP:5-FAM-AGATCGCGGTCTCCTGTCCA-TAMRA斗正义引物COG臼2F瓦:5-C反义引物COG2R:5而丁CGACGCCA丁CTTCAT丁CACA-
8、3探针R孙G2-TP:5人FAM伽TGGGAGGGCGATCGCAATCT-TAMRA-36. 1 实时荧光PCR仪。6.2 PCR仪。6.3 电泳仪。6.4 凝胶分析成像系统。6.5 冷冻离心机。6.6 勾浆器。6. 7 水浩锅。6.8 徽章可调移液器。6.9 高压灭菌锅。6.10 -800C低温冰箱。2 326 扩增片段大小/bp107 119 SN/T 1635-2005 6. 11 50 mL离心管。6. 12 元RNase玻璃容器z见附录A.2.L6.13 元RNase离心管(1.5 mL, 15 mL)、无RNase移液器吸嘴(20L、200L、1000L、元RNase药匙、二IG
9、RNase PCR薄壁管z见附录A.2. 20 6.14 1. 5 mL磁性抽提架。7 检击tl方法7. 1 实验案要求实验室设施应达到SN/T1193实验室技术要求。7.2 检甜步骤7.2.1 病毒的富集7.2. 1. 1 解剖取下贝类的中肠腺组织。7.2. 1.2 取5g中肠腺组织加入35mL甘氨酸缓冲液(pH9.5)。7.2. 1.3 匀浆器高速匀浆3min，将匀浆被装人50mL离心管，370C混育30min或室温下振荡30min, 40C ,10000 g离心30mino 7.2. 1. 4 移取上清至一50mL新管，加入等体积的PEG8000溶液，颠倒5次混匀。冰上放置至少1 h ,
10、40C ,10 000 g离心5min，弃去上请，保留沉淀。7.2.2 病毒RNA的提取和纯化7.2.2. 1 为防止RNA降解，所用实验用具及溶液应无RNA酶，操作过程中应自始至终佩戴抛弃式橡胶或乳胶手套，并经常更换，以避免将皮肤表面的RNA酶污染用具或带入溶液。7.2.2.2 在7.2.1.4的沉淀中加入5mL Tri而reagent或其他等效裂解液，剧烈震荡30s，室温放置5 mino 7.2.2.3 转移洛液人一15mL元RNA酶离心管，加人1.2 mL二三氧甲:境，剧烈震荡30s，室温放置5 min,4.C ,12000 g离心5min，吸取上清至另一15mL元RNA酶离心管。7.2
11、.2.4 在上清液中加入0.5倍体积约2.5mL)的异丙酶，室温放置5min,4.C , 5000 g离心5mino 7.2.2.5 弃去上请，用5mL，4.C预冷的75%乙醇洗涤沉淀。7.2.2.6 沉淀章悬于300L元RNase的水中，将悬液转移至一1.5 mL无RNase的离心管中。7.2.2.7 加入400L1X RNA吸甜缓冲液，麓荡30s，60.C放置3mino 7.2.2.8 加入100LDynabeads-oligo(dT)2S轻柔混合，磁性抽提架上放置1mino 7.2.2.9 弃去上清，加入500L2XRNA吸陆缓冲液，室温下晃动5min洗涤。7.2.2.10 离心管在磁性
12、抽提架上放置1min，弃去上清。加入500L洗液，颠倒5次混匀，离心管在磁性抽提架上放置1min，弃去洗滚。重复洗涤3次。7.2.2. 11 沉淀用100L元RNase水悬浮，90.C放置2min释放RNAo磁性抽提架上放置1min，取上清液。将上清移至另一1.5 mL无RNase的离心管中，加入20U RNase抑制剂，即时进行R下PCR检测。7.2.3 普通RT-PCR检测7.2.3. 1 普通RT-PCR反应体系检翻tl贝类中诺沃克病毒的一步法普通RT-PCR反应体系见表3。每个反应体系设置两个平行反应。反应体系中各试剂的蠢可根据具体情况或不同的反应总体积进行适当调整。也可采用RT-PC
13、R两步法试剂盒。以诺沃克病毒RNA作为阳性对照，以不含有诺沃克病毒的贝类RNA作为阴性对照，以水代替模板作为空白对照。3 SN/T 1635-2005 表3普通RT-PCR反应体系名称贮液浓度终浓度加样最/LRT-PCR缓冲液5X 1X 10 MgS. 25 mmol/L 1 mmol!L 2 dNTPs 10 mmol/L 0.2 mmol/L 1 正义引物50 p.mol!L 1mol/L 1 反义引物50 p.mol!L 1mol/L 1 逆转录酶5 U/L 0.1 U/L 1 DNA聚会酶5 U/L 0.1 U/L 1 模板10 水(元RNase)23 总体积50 一一一7.2.3.2
14、 普通RT-PCR反应参数普通R丁子CR的反应参数:42.C，60min; 94.C , 10 min; 94.C , 1 min, 37.C , 90 s , 74.C , 1 min, 40个循环;74 .C ,7 min. 7.2.3.3 PCR产物的琼脂糖凝胶电珠检j将适最50XTAE稀释成1X丁AE溶液，配制澳化乙键含量为0.5g/mL的1.5%琼脂糖凝胶。取15LPCR产物，加1.5L上样缓冲撞点样进行电泳，并加DNA分子量标记点样以判断PCR产物的片段大小。电压大小根据电泳槽长度来确定，一般控制在3V/cm5 V/cm长度，当澳酷蓝移动到凝胶边缘时关闭电源，电泳检测结果用凝胶分析
15、成像系统记录。若样品检测出现预期带型，则使用DNA分析系统对PCR产物进行测序，并与GeneBank数据库中的序列进行比对。7.2.4 实时荧光RT-PCR7.2.4.1 实时荧光RT-PCR反应体系检甜贝类中诺沃克病毒的一步法实时荧光RT-PCR反应体系见表4。每个反应体系设置两个平行反应。反应体系中各试剂的最可根据具体情况或不同的反应总体积进行适当调整。可采用商业RTPCR一步法或两步法试剂盒。以诺沃克病毒RNA作为阳性对照，以不含有诺沃克病毒的贝类RNA作为阴性对照，以水代替模板作为空白对照。表4实时荧光RT-PCR反应体系力日样最/L名称贯主液浓度终浓度GI型病毒GII病毒RT-PCR
16、缓冲液5X 1X 10 10 且在gS.25 mmol!L 1 mmol!L 2 2 dN丁Ps10口rmol/L0.2 mmol/L 1 1 正义引物50mol/L 1mol/L 1 1 反义弓l物50mol!L 1mol/L 1 1 逆转录酶5 U/L 0.1 U/L 1 1 DNA聚合酶5 U/L 0.1 U/L 1 1 一一一一一一一一一一4 SN/T 1635一2005表4(续)加祥量/L名称贮液浓度终浓度GI裂病毒GII病毒探针5mol/L 0.1mol/L 探针a:31 探针h:1模板10 10 7j(元RNase)19 22 总体积50 50 7.2.4.2 实时荧光RT-PC
17、R反应参数实时荧光RT子CR的反应参数为:48.C, 45 min;50.C , 2 min;95.C ,10 min;95.C , 15 s , 56.C ,1 min , 45个循环。注:不同仪器根可根据仪器要求将反应参数作适当调整。8 结果判定及表述8. 1 结果如i定8. 1. 1 普通RT-PCR对样品进行RT子CR检测，如果阴性对照和空白对黑未出现条带，陆性对照出现预期大小的扩增条带，而样品未出现预期大小的扩增条带，则可判定样品诺沃克病毒阴性。如果阴性对照和空白对照未出现条带，陆性对照和样品出现预期大小的扩增条带，并且对PCR产物序列分析证实待现样品的序列与诺沃克病毒的cDNA序列
18、一致，则可判定样品诺沃克病毒阳性;若两者序列不一致，则可判断样品诺沃克病毒阴性。8.1.2 实时荧光RT-PCR检测样品的Ct值大于或等于45时，则判定诺沃克病毒阴性。检测样品的C，值小于或等于30时，则判定诺沃克病毒雨性。检测样品的c.值小于45而大于30时，应重新进行测试，如果重新测试的C，值为大于或等于45时，则判定诺沃克病毒阴性;如果重新测试的Ct值小于45，则判定诺沃克病毒雨险。8.2 结果表述诺沃克病毒阳性。诺沃克病毒阴性。5 SN/T 1635-2005 捕录A(规范性附录溶霞的配制A.1 普通溶溜的配制A. 1. 1 甘氨酸缓冲液z含0.1mol/L甘氨酸，0.3mol/L N
19、aCl, pH 9.5 甘氨酸7.5 g 氟化铀(NaCl)双蒸水5 mol/L氢氧化铀溶液17.5 g 800mL 调pH至9.5加双蒸水至1000 mL, 121.C , 15 min灭菌备用。A. 1. 2 PEG 8 000溶破:含16%(w/v)PEG 8000,0.525 mol/L NaCl PEG 8000 16 g 氧化铀(NaCl)3. 07 g 加双蒸水至100mL, 121.C ,15 min灭菌备用。A. 1.3 50XTAE缓冲液A. 1.3.1 0.5 mol/L EDTA个1a2(二水乙二镀四乙酸二铀溶液，pH8.0EDTA-Na2 .2日20灭菌双蒸水5 mo
20、l/L氮氧化锅溶液186.1 g 800 mL 调pH至8.。灭菌双蒸水如至1000mL, 121.C , 15 min灭菌备用。A. 1.3.2 丁AE电泳缓冲液(50X)配制j起基ftI基氨基ftI烧(Tris)冰乙酸242 g 57.1 mL O. 5 mol/L EDT A溶液，pH8.0100 mL 灭商双蒸水加至1000 mL, 121.C ,15 min灭菌备用。用时用灭菌双蒸水稀释至1X使用。A. 1.4 澳化乙键(EB)溶液(10g/p.L)漠化乙键20 mg 灭菌双蒸水20 mL A. 1.5 含0.5g/mL澳化乙链的1.5%琼脂糖凝胶的配制琼脂糖1. 5g 1XTAE电
21、泳缓冲液加至100mL ?昆合后加热至完全黯化，待冷至50.C55.C时，加澳化乙键(EB)溶液5L，轻轻晃动摇匀，避免产生气泡，将椅子置入电泳槽中，然后将琼脂糖溶液倒人电泳板上，待凝固后(需约40min) ，取下梳子，备用。A. 1.6 10X加样缓冲液6 聚蔚糖灭菌双蒸水澳酣蓝二甲苯青25 g 100 mL 0.1 g 0.1 g SN/T 1635-2005 A.2 RNase的去酷和无RNase溶涩的配制配制溶被用的酒精、异丙障、Tris、EDTA、LiCl、浓HClNaOH等应采用未开封的新品。配制溶液所用的超纯水、玻璃容器、移被器吸嘴、药勺等塑料用具应无RNaseo操作过程中应自始
22、至终佩戴抛弃式橡胶或乳胶手套，并经常更换，以避免将皮肤上的细商和真菌以及人体自身分泌的RNA酶污染用具或带人溶液。A. 2.1 玻璃容器应在2400C烘烽4h以去除RNase。A.2.2 离心管、移被器吸嘴、药勺等塑料用具应用0.01%的DEPC(焦碳酸二乙醋)水室温浸泡过夜，然后灭菌，烘干;或直接踊买元RNAase的相应规格离JL管、移液器吸嘴。A.2.3 元RNase超纯水超纯水100 mL 焦碳酸二乙醋(DEPC)50L 室温过夜，1210C，15min灭窟，或直接购买无RNase超纯水。A.2.4 75%乙薛元水乙黯无RNase超纯水现配现蹄。A.2.5 1XRNA吸甜缓冲被A. 2.
23、 5. 1 1 mo1/L Tris-HCl pH7. 5 Tris 无RNase超纯水36.5%盐酸1 mo1/L盐酸7.5 mL 2.5 mL 1. 21 g 6 mL 0.75 mL 调pH至7.5加元RNase超纯水至10mL，分装到1.5 mL元RNase离心管中，一20.C保存。A. 2. 5. 2 O. 5 mol/L EDT A白Na2(乙工按图乙酸二铀), pH7. 5 EDTA-Na2 .2H20 元RNase超纯水10皿o1/L氢氧化铀1. 86 g 6 mL i题pH至7.5加元RNase超纯水至10mL，分装到1.5 mL元RNase离心管中，一20.C保存。A. 2
24、. 5. 3 5 mo1/L LiCl LiC1 2.12 g 元RNase超纯水8 mL 加元RNase超纯水至10mL，分装到1.5 mL无RNase离心管中，一20.C保存。A. 2. 5. 4 1 X RNA吸附缓冲液z含20mmo1/L Tris-HCl (pH7.日，1. 0 mo1/L LiCl, 2 mmo1/L EDTA-Na2 ,pH7. 5 1 mo1/L Tris白日ClCpH7. 5) 5 mo1/L LiCl O. 5 mo1/L EDT A叩Na2(pH7. 5) 元RNase超纯水总体积现配现用。200L 2000L 40L 7 760L 10 mL A.2.6
25、 2XRNA吸附缓冲液z含40mmo1/L Tris-HCICpH7. 5) , 2.0 mo1/L LiCl , 4 mmo1/L EDTA而Na2 ,pH7. 5 7 SN/T 1635-2005 1 mol/L Tris-HCl CpH7. 5) 5 mol/L LiCl O. 5 mol/L EDT ANa2CpH7.5) 无RNase超纯水总体积现配现用。400L 4000L 80L 5 520L 10 mL A.2.7 洗液z含10mmol/L Tris-HCICpH7.日，0.15mol/L LiCl,l mmol/L EDTANa2 ,pH7. 5 8 1 mol/L Tris
26、-HCl CpH7. 5) 5 mol/L LiCl O. 5 mol/L EDT A而Na2CpH7. 5) 无RNase超纯水总体积现配现用。100L 300L 20L 9 580L 10 mL SN/T 1635 2005 Foreword Annex A of this standard is normative annex. This standard was proposed bCertification and Accreditation Administration of the People s Re public of China (CNCA) .丁hisstandard
27、is under the jurisdiction of CNCA. This standard was drafted by Guangxi EntryExit Inspection and Ouarantine 8ureau of People s Re public of China and Shanghai Entry-Exit Inspection and Ouarantine 8ureau of People s Republic of China. Main drafters of this standard are Liu Junyi , Pan Liangwen , Zhan
28、g Shuya , Li Xiaohong , Wei Meil iang , Luo Zhaofei. This standard is a professional standard of ent印刷exitinspection and quarantine promulgated for the first time. 9 SN/T 1635-2005 Detection of norovirus in shellfish一conventional RT唰PCRand real-time RT幽PCR1 Scope 丁hisstandard specifies conventional
29、RT-PCR and real-time RT平CRmethod for the detection of noro virus in shellfish. This standard is applicable to detection of norovirus in shellfish. 2 Normative references 丁hefollowing normative document contains provisions which, through reference in this text, con stitute provisions of this standard
30、. For dated references , subsequent amendments to, or revision of, anof these publications do not appl. However, parties to agreement based on this standard are encouraged to investigate the most recent editions of the normative document referred to ap plies. For undated references , the latest edit
31、ion of the normative document referred to applies. SNjT 1193 Technical requirements of gene analysis laboratory 3丁ermsand Definitions For the purposes of this standard , the following terms and definitions appl. 3. 1 Real-time fluorescence RT-PCR: Real-time fluorescence RT唰PCRis set up on the basis
32、of conventional R丁平CRbadding a specific probe.丁hisprobe is composed of a short (ca. 20-25 bases) oligodeoxnucleotide that is labeled with a reporter dye on the 5 terminus and a quenching dye on the 3 terminus. When the probe is intact, energtransfer occurs between the two fluorophors and emission fr
33、om the reporter is quenched by the quencher. During the extensionphase of PCR , the probe is cleaved by 5 nuclease activity of Taq polymerase, thereby releasing the reporter from the oligonucleotide幡quencherand producing an increase in reporter emission intensit. Real-time PCR allows for the direct
34、detection of the PCR product during the exponential phase of the reaction , combining ampl_ification and detection in one single step. 3. 2 Ct. value , cycle threshold : 丁heThreshollline is the level of detection or the point at which a reaction reaches a fluorescent in-10 SN/T 1635一2005tensity abov
35、e background. The ccle at which the sample reaches this level is called the cycle thresh old, Ct. 4 Summary of the Method Viral RNA is extracted by using Tri-reagent or other equivalent Iysis buffer,and is further purified b magnetic poly dT beads as there is a PolCA) at 3 end of the norovirus RNA.
36、Purified RNA is detec ted by conventional R下PCRor real-time R下PCR.When doubtful , the PCR product should be co怀firmed bsequencing. 5 Reagents AII reagents for experiment should be analtically pure. Unless otherwise stated , water for experi ment should be distilled water or deionized water. 5. 1 Spe
37、cimen positive for norovirus: Afforded by the department designated bState Administra-tion of Quality Supervision. Inspection and Quarantine. Store in - 800C refrigerator. 5. 2 Glycine buffer: See annex A. 1. 1. 5. 3 PEG 8 000 solution: See annex A. 1. 2. 5.4 Lsis buffer: Tri-reagent or other equiva
38、lent Iysis buffer: 5.5 PolC dT) beads: Dnabeads-oligoCdT)25 or equivalent products. 5.6 RNase-free water:See annex A. 2. 3. 5.7 75% ethanol:See annex A. 2. 4. 5. 8 Isopropanol : newly sealed. 5. 9 1 x RNA binding buffer: See annex A. 2. 5. 5.10 2 X RNA binding buffer:See annex A. 2. 6. 5.11 Washing
39、buffer:See annex A. 2. 7. 5.12 RNase inhibitor. 11 SN/T 1635-2005 5.13 Reverse Transcriptase AMV. 5.14 DNA polymerase. 5.15 dNTPs mixture:dATP,dUTP,dCTP,dGTP 10 mmol/L each. 5. 16 Primers and probes: Primers and probes are synthesized according to the sequences of table 1 and table 2 and each is dis
40、solved in RNase-free water to a final concentration of 50mol/L. 5.17 DNA marker: 100 bp-2 000 bp. 5. 18 50 x T AE buffer: See annex A. 1. 3. 5.19 Ethidium bromide solution(10g/L) : See annex A. 1. 4. 5.20 1.5% Agarose Gel containing 0.5g/mL ethidium bromide:See annex A. 1.5. 5.21 10 x loading buffer
41、:See annex A. 1.6. Table 1 Primers for conventional RT-PCR Virus group sequences of primers Length of the expected detected PCR product/bp GI ,Gl sense primer JV12:5-ATACCACTATGA丁GCAGATT A-3 326 antisense primer JV13:5人了CATCATCACCATAGAAAGAG-3-一一一-一一一一一-一一-Table 2 Primers for real帽timeRT-PCR Virus gr
42、oup Length of the sequences of Primers and probes expected PCR product detected /bp Sense primer COG1 F:5 -CGYTGGAiGCGNTTYCATGA-3 GI Antisense primer COG1日:5-CT丁AGACGCCATCATCATTY AC-3 107 Probe RING1 (a)-TP:5-FAM-AGATYGCGATCYCCTGCCA幡AMRA-3Probe RING1(b)-丁P:5-FAM帽AGATCGCGGTCTCCTGTCCA帽TAMRA-3Sense pri
43、mer COG2F: 5人.CARGARBCNATGTTYG只TGGATGAG-3Gl Antisense primer COG2R: 5 -TCGACGCCATCTTCA TTCACA-3 119 Probe RING各TP:5 -FAM-TGGGAGGGCGATCGCAA TCT-T AMRA-3 一6 Instruments and Apparatus 6. 1 Real time fluorescent PCR cycler. 12 SN/T 1635一20056.2 PCR amplifier. 6. 3 Electrophoresis sstem. 6. 4 Electrophor
44、esis documentation and analsis sstem. 6. 5 Refrigerated centrifuge. 6. 6 Blender. 6.7 Temperature controlled water container. 6.8 Adjustable micropipettor. 6. 9 Autoclaves. 6.10 - 800C refrigerator. 6. 11 Centrifge tubesC50 mL). 6. 12 RNase-free glass container: See annex A. 2: 1. 6.13 RNase-free ce
45、ntrifuge tubesC1. 5 mL, 15 mL), pipette tipesC20L，200L，1 000L) ,medicine spoons , thin wall PCR tubes: See annex A. 2. 2. 6.14 1.5 mL magnetic bead attractor. 7 Detection Method 7.1 Lab req川rementEstablishment of the testing laboratory should be accord with technical requirements of SN/丁1193.7.2 Pro
46、cedures 7.2.1 Virus concentration 7.2.1.1 Dissect the stomach and digestive diverticula from the whole shellfish. 7.2.1.2 Homogenize up to 5 9 tissueCstomach and digestive diverticula) in 35 mL glycine buffer. 7.2.1.3 Blend the mixture for 3 min at high speed by using a laboratory blender. Transfer
47、the ho-13 SN/T 1635-2005 mogenate to a 50 mL centrifuge tube. Incubate homogenate for 30 min at 370C or shaking for 30 min at room temperature. Centrifuge homogenate at 10 ,000 X 9 for 30 min at 40C . 7. 2. 1. 4 Transfer the supernatant to a clean 50 mL tube. Add equal volume of PEG 8 000 solution t
48、o the supernatant and invert 5 times to mix. Place the tube on ice no less than 1 hour. Centrifuge the tube at 10 ,000 x 9 for 5 min at 4C and save the PEG pellet. 7.2.2 RNA extraction and purification 7.2.2.1 AIl equipment and solution should be RNase-free to prevent PNA degradation. Wear dis posab
49、le latex gloves and change them frequently to prevent the introduction of RNase from skin sur faces throughout all steps. 7.2.2.2 Resuspend the pellet of 7. 2.1.4 in 5 mL Tri-reagent or other equivalent Iysis buffer, ver tex 30 sec , incubate for 5 min at room temperature. . 7.2.2.3 Transfer to a 15 mL RNase唰freecentrifuge tube, add 1. 2 mL chloroform, vertex 30 sec
