实验动物 近交系小鼠、大鼠SNP标记检测法》征求意见稿.docx

附件4ICS65.020.30B44中华人民共和家标准GB/T××××××××实验动物近交系小鼠、大鼠SNP标记检测法1.aboratoryanimal-Singlenucleotidepolymorphismsmarkermethodsforinbredmiceandrats（征求意见稿）XXXX-XX-XX 实施XXXX-XX-XX发布国家市场监督管理总局国家标准化管理委员会GB/T××××××××-I-X-1刖百本标准按照GB/T1.1-2009的规则起草。本标准由全国实验动物标准化技术委员会(SACzTC281)提出并归口。本标准起草单位：中国食品药品检定研究院，上海实验动物研究中心、陕西省中医药研究院、山西医科大学、广东省实验动物监测所本标准起草人：魏杰、王洪、岳秉飞、赵莹、李欢、周佳琪、赵丽亚、汤建平、姚养正、宋国华、王静、范春、陈国强GB/T××××××××实验动物近交系小鼠、大鼠SNP标记检测法1范围本标准规定了实验动物近交系小鼠、大鼠单核甘酸多态性(SNP)标记检测方法及判断标准。本标准适用于实验动物近交系小鼠、大鼠遗传组成分析、品系鉴别和质量控制。2规范性引用文件下列文件对于本标准的应用是必不可少的。凡是注明日期的引用文件，仅注日期的版本适用于本标准。凡是不注日期的引用文件，其最新版本(包括所有的修改单)适用于本文件。GB14923-2022实验动物遗传质量控制3 术语和定义下列术语和定义适用于本规程。3.1SNP标记singlenucleotidepolymorphismsmarker单核甘酸多态性标记，是指在基因组水平上由单个核甘酸在位置上发生转换、颠换、插入或缺失等引起的DNA序列多态性，为基因分析的第三代分子标记。常见的SNP以二等位多态性为主。3.2纯合性homozygosity同源染色体的相对位置上具有相同基因的状态。近交系动物通过连续的近亲交配绝大部分的位点都具有纯合性。一个品系内任何个体问进行交配产生的后代也具有纯合性。3.3同基因性iSOgenicity一个近交品系中所有个体在遗传上是同源的。如对近交系动物的基因进行检测，一个品系内不同个体的基因型完全一致。3.4个体性indiViduaIity就整个近交系动物而言,每个品系在遗传上都是独特的，大多数近交品系可通过各自的SNP遗传概貌相互区别。4 主要试剂与仪器4.1 试剂DNA提取试剂盒，PCR反应及产物纯化试剂盒，测序反应试剂盒、琼脂糖。4.2实验设备和器材4.2.1仪器设备：电泳仪，离心机，PCR仪、紫外凝胶成像系统、遗传分析仪，水浴锅、微波炉、混匀振荡器。4. 2.2器材：剪刀、镜子、采血管、移液器、离心管、PCR管、琼脂糖制胶板。5. 方法原理小鼠和大鼠基因组内均存在数量极为丰富的SNP位点。通过筛选品系特异性的SNP标记位点，并在该变位点上下游设计引物，通过PCR扩增、连接酶反应、测序、毛细管电泳等分子生物学方法分析，得到各近交系的遗传概貌，定期对他们进行质量监测。6位点选择6. 1近交系小鼠检测（直接测序法）常规近交系小鼠从表1中前27个SNP标记进行PCR扩增，使用直接测序法进行检测。除表1前27个标记外，C57BL/6亚系需要加测表1中28-32号SNP标记，BLBc亚系需要加测表1中33-35号SNP标记。各实验室可根据自身条件设计或选择引物。表2中引物和退火温度可供参考。其中，产物长度为约数，不作为判别标准。退火温度为推荐退火温度。实际操作中，可根据各实验室具体条件进行优化调整，经比对验证，58°C-60C均可得到目的片段。表1近交系小鼠35个SNP标记检测位点序号SNPID序号SNPID序号SNPID序号SNPID1rs302279610rs302317719rs302338228rs37096242rs302282511rs302320320rs309104829rs36597873rs302288312rs302322621rs309117430rs37223134rs302295313rs308998422rs302343631rs37021585rs302297714rs308910923rs302344232rs37248766rs302303415rs308867324rs302345033rs37126927rs302303916rs302325125rs308934934rs37060828rs302306417rs302334226rs302348135rs36568019rs302316218rs302338127rs3089604/表2近交系小鼠35个SNP标记引物及退火温度No.primersequenceProduct(bp)TmCOChr.1rs3022796-FGtatggtggggagagcacac344611rs3022796-RGagtcaggtccaaccaaccc2rs3022825-FGgcactgcagtattagccga299601rs3022825-RCctggcatgtactgtgaggc3rs3022883-FAgktgggggaggaaccctg209602rs3022883-RAggcaggcatcatacattgga4rs3022953-Frs3022953-RCatttgacaaggtatgcagggcGtattttgtaggggcaccaga1985935rs3022977-FAtgagaggaacaacaagcacgt385594rs3022977-RCcccaagatttctacaccaaag6rs3023034-FTGAGTTnCACTCTTGCTGGC249605rs3023034-RAgcccttctgatagttgaacgac7rs3023039-FCagccataccactctgctcc459615rs3023039-RGgtcagaacccatgtccagg8rs3023064-FGcccagccagtgggtc153586rs3023064-RTggcaataattgtatcttgtgccc9rs3023162-FTtttcactgtgttcccgtgac262607rs3023162-RAgacctaacagggaacaggca10rs3023177-FTTTGAGTTGCAGGGTTnCCTG200598rs3023177-RGcctttacagccaaagacttatca11rs3023203-FGgtctcttggcatctggcat183589rs3023203-RGagcttgtagagagaacccct12rs3023226-FGtgacaaccctcctggtgta185599rs3023226-RAactccgtgtggggtagtga13rs3089984-FGcctaacatcactttttgggagc1996010rs3089984-RGgagcaattagaatcccaggtg14rs3089109-FTAACAAAGCTCCACTCCTCAAG1915910rs3089109-RAatgggtctgtcagagtktctca15rs3088673-FAccattcttctcaactaagtctgct1905911rs3O88673-RTggtgatagtgatgggctct16rs3023251-FTcagcatctcccttaaccttctt1866011rs3023251-RAccagctcctagtgatgaca17rs3023342-FTgactggaccgtaacgagag1986012rs3023342-RCaggccagaccgtgctaatg18rs3023381-FGaggtagagatgcagcaggg1695913rs3023381-Rggagggatggatggtggaga19rs3023382-FAggagccgctggttctgata1895913rs3023382-RAtgtacacatgagactccggc20rs3091048-FGctatagaccaggagttgccttt1856014rs3091048-RTggacaagcaccctcacaata21rs3091174-FTaaaatggtcctgcattttgcca1985915rs3091174-RTactcacatacaactgaacaaggca22rs3023436-FAacttacgatgtggggtgcaa1986016rs3023436-RAGATGCACGnCACACTGAT23rs3023442-FAgcggaggtgcttgttacat2976017rs3023442-RAacacccaaatgccagccta24rs3023450-FAtggatcagggaagagcacc2166017rs3023450-RCtgcctgcatcccctaactc25rs3089349-FCcctgtgctcatggtgtagaaa2996118rs3089349-RTcagggtggatgatgtccaaag26rs3023481-Frs3023481-RTctgggccactactttcacttcCAGCACTTTCCACAGnGGG250601927rs3089604-FTtttcagggccaaagcagact29060Xrs3089604-RTcctcctaactaagtccccctttt28rs3709624-FAcagtgctggcttgtggtagag673618rs3709624-RCgaakatgaccgagaagttga29rs3659787-FCcacaacaaagagccagaccaa2775911rs3659787-RTaggcttggccttcatctaagc30rs3722313-FGtgtctctcaccaggacagc2726013rs3722313-RAACGGGTCGTAGAACnGGC31rs3702158-FActctccaaagaggaacagctt2365915rs3702158-RTacagagtaccctagcacgcc32rs3724876-FAAGTGTCAGTTTTAnGTTTCCAGT1775819rs3724876-RCaagttccckttggctggc33rs3712692-FAgggcccgattgtagtgttta197591rs3712692-RCctgtgaagtgaatgagcttgc34rs3706082-FAcagcgggaattggatggtat241604rs3706082-RGtgatcaccacactcttccgt35rs3656801-FAcccacgagaacaatgaggc1635918rs3656801-RGgtagggtcagcactggaac6.2近交系大鼠检测（LDR法）21个SNP位点共分为2组进行PCR-LDR,表3、表4分别为21个SNP位点的引物和探针，覆盖大鼠的20条常染色体和X染色体。表321个近交系大鼠SNP位点引物的扩增条件和染色体分布组别SNP位点引物序列染色体MgC12(mmolL)退火温度（Drs65970293TGCACATTGGGACTGCTTrAAtctggcatggacagagctt1rs106996961AtagggccaaggtcccattcGaaaagaacgctaatcacaggaca2rs63860341CaatgaatcaaaggcaaccaCcacggtccctatgtgaact31rs64750237CATATGGCTTTGGAGGAAGttttcagctggagctgtcc4359rsl07261116CagcattggcaagtcttcctTgataaaaggagcagcagca5rs64102787CggggatcaaagagacaaaaAtagtgccaaagcccaacac6rs105187832AgactactgggctgctcaccCATGGCACACCTTTCTTTCA7rsl06114574Ggccccctaatttccataaa8MgC12退火温度组别SNP位点引物序列染色体(mmolL)(C)TccaaacatctctggtgtgcGctggaagtctctggagtggrs64682441Acaagtggactggctcaggt9Atccctctctttgccaggatrs65067166Gtagggtgaggctgcttctg10Accccctcttgctttctgttrs8167985Ctgctcttggatgaaaacca11Gcccttcccaaggtcatactrs106672258Aaagcacaaaaatgccatcc12Aacctggtgatttgtcagagcrs65921305Ggcagactccaggaagattg13Attagccacggatgaagtggrs106335590Tttgctgtctggctgatgac14AgagcagctgccaaaatagcrsI()6458396Aaacctggctttagcaagca15Tgtgaagaacacaggctctccrs106888236Tgccaaacaccatgtaatgaa162rs63814610Taggacagggggaacaactg17359CccaagcatccttgactgttCgtgtcattcctgactgtggrs8152541Acaggagctggcactgaatc18Ttctgcaatcaagggtggatrsl05824121Ggccaccaaagatacacagg19Tacgccattttgcgacattarsl3453252Ggttccctacgaggaacgac20Catcccagaagccctgtttare65108991GaagtcagtgctgtgcctcaX表421个近交系大鼠SNP位点探针的序列组别位点名称探针序列rs65970293.modifyGAAAGCAcACArAlAAGAAGl,l,l,l,ltltttttttttttttrs65970293_Tttttttttttttttt7AGAAGCTATCCCAGTGTTATGGACArs65970293_CtttttttttttttttTTTAGAAGCTATCCCAGTGTATGGACGrs10699696l-modifyACTATTTACAGAArGGUACCl1111111Utttttttttttttrsl06996961_GIttttttttttttttTnTGAAAAATTTATTCACAAAATAAACrsl06996961_TtttttttttttttttTTTTTTGAAAATTATCACAAAATAAAArs6386034LmodifyTGACTGTTGTTCCGAATTACITTTTTTTTTtttttttttttttttrs6386034l_Ctttt11tttttttttincciggcgacIcaagccc11CACCGrs6386034I_TttttttttttttttiriririciciGgcgactcaagcccttcaccars64750237-moditygAAaaacAcAc1GGgaa1gi1111111l1ItUtttttttttttt组al位点名称别探针序列rs64750237_Ttttttttttttttttrr,Tigaaggcttaaagaggataaagga>64750237-CtttttttttttttttTTTTTTTTTTTGAAGGCTTAAAGAGGATAAAGGGrsl07261116_modifyralO7261116-GTAAAccCTTGCTGTTCCACTTTTTTTTTTTTTTTltttttttttttttttttttmttttttt11rr11111CcttaagaaaaaaaaaactgttctcrslO7261116_CtttttttttttttttTTTTTTTTTTTCCTTAAGAAAAAAAAAACTGTTCTGrs64102787.modifyrs641O2787.CCAGTClAGACCIClGCclCc111rnriillIttttttttttutttIttttttttttttttiin111rn1CT11ACCCCGCCTGTCAGCTCCCAGre64102787.AtttltttttttttttH11H1CllTACCCCGCCTGTCAGCTCCCATrsl05l87832_modifyrslO5187832_GCcCAAGCGGTGAGCAGCCCATTTTTTTTTTTTTTTTTTttttttttttttttttttttttttttttttTTTTTTTTTTTTTAGCTACTTCCGTTTATGAATAAACCrslO5l87832.TIttUttttttttttTTTTTTTTnTTTTTAGCTACTTCCGTTTATGAATAAACArsl06II4574.modifyrslO6H4574.TAAGCAGTATACAGTCTGGAGTTTTTTTTTTTTTTTTTTTTttttttttttttttttttttttttttttttTTTTTTTTTTTTTTTTCCCACTTCCTTACTATTTGACTCArs106114574.Atttttttttttttttrrn1111l1l1CCCACTTCC11AC1A1GACC1rs64682441.modifyrs64682441.TATTAGCAGAGAGTAAGACAGTTTTTTTTTTTTTTTTTTTTTTttttttttttttttttttttttttttttttrrniiiiniiirnIcatatcccaaatagcaaggctcagars64682441.Ctttttttttttttti1i11ill1IlTllIIlCAlAlCCCAAAlAGCAAGeiCTCAGGrs65067166-modifyrs65067166.GCAAGTCATCTATGCTCT1C11111111ill111111111111ItttttttttttttttIttttttttttttttirn1ITlllIlTllllIICCAAGeTACeTCcTTCACArCCAGCre65067166.CUttutttttttttrnriiiin111rnirlCcAAGC1aCu1CellcAcACcAGgrs8167985.modifyrs8167985.TCACAaAICCAGaA1j1CCC111ill111lLl1H111Llttttttttttttttttttttttttttmtrnlll11IliHl111GAGAAAGGAATAATTGGAGGTTACArs8167985.GUtttttttttttttirniiirniiiruAgaaaGgAaiaahGgaGuiaCCrs106672258-modifyrs106672258_TTGGGTAGGATATTGGAGTTTTTTTttttttttttttttttttttttttttttttTAGCCAGCCCCCAGACACCTCGATArsl06672258_CttttttKtttttttTTTAGCCATGCCCCCAGACACCTCGATGrs65921305-modifyn;65921305_TGGGAAAAATAGATTTGCAGTTTTTTTTTtttttttttttttttttttttttttttUtTTTT(mTCATTGAATGTTGAATTACA11,659213O5-Gtttttttttttttttl'1"1TlIClIlCAlIGAAlGriGAAlllACCrs106335590_modifyrsl06335590_GTGTTAATAAAACAAACATATTTTTTTTTTTtttttttttttttttMttttutttttttTmTAGAAAGAAcAGACTCACTCAATTCCrs10633559(LTtttttttttttttttTTTTTTTAGAAAGAACAGACTCACTCAATTCA2rs106458396_modifyrsl06458396_T1CCA1111IAiCCClGGlll1111illIrittttttttttttttttttttttttttttttTTTTTTTAAACCTGGCTTTAGCAAGCAATGTArsl06458396_CtttttttttttttttTTTTTTTTTAAACCTGGCTTTAGCAAGCAATGTGrs106888236TIodifyrslO6888236_CGGGATGCTAAAGGTTTCTGATTTTTTTTTTTTTTtttttttttttttttttttttuttttuti'iirmIiagtctgtacaacagtatcaacgaag11>1O6888236.AtttttttttttttttTTTTTTTTTTTAGTCTGTACAACAGTATCAACGAATrs63814610-modifyn;6381461(LCGTGCTGGGCCTTGTGACCTTTTTTTTTTTTTTTTTTtttttttttttttttttttttttttttUtTTTTTTTTTTTCAGGCACTGAGGTAGGTACCACG,63814610.Ttttttttttttttttn11rn11111CaGGCaC1GAGGiaGGiACCaGGCArs8152541.modifyGTrAGAGGTGACAGGAGTGmTr11rIT门TTTmttttttttttttttt组al位点名称别探针序列n;8152541_Gttttttttttttutl"11,l'l11llH,m,CCTGAGGACACTGCATCACACTCre8152541_AtttttttttttttttTTTTTTTTTTTTTTTTCCTGAGGACATCTGCATCACACTTrs105824121.modifyGTGccTcccCACCCCTAACATTTTTTTTTTTTTTTTTTTTtttttttttttttttrs105824121.Atttttttttttttttiihi11rnrr11acag11,Igaitagttaagaaccttn;105824121_Gtttttttttttttttm1111H111inIAiCAGrrTGATTAGTTAAGAACCTCrsl3453252_modifyGTAGTGTACCTGGTAGAAGATTTTTTTTTTTTTTTTTTTTTTtttttttttttttttrsl3453252_Gttttttttttutttiin111rn1IirniuGIlCCC1ACGAGGAACGACGCIGCn;13453252_CUttttttttmttrnri1111111riill1GGTTCCCTACGAGGAACGACGCTGGrs65108991.modifyTAfrATAAACAAAGCTTGGAiinrr11rn1rrrrnIirnIttuuttttutttrs65108991.AtttttttttttttttTTTTTTTTTTTTTTTTTTTAAGTCAGTGCTGTGCCTCAGTCTATrs65108991_CIttttttttttttttTTTTTTTTTTTTTTTnTTTTAAGTCAGTGCTGTGCCTCAGTCTAG6.3近交系大鼠检测（直接测序法）常规近交系大鼠从表5中前31个SNP标记进行PCR扩增，使用直接测序法进行检测。研究大鼠免疫遗传标记，可加测表5中32-38号SNP标记。各实验室可根据自身条件设计或选择引物。表6中引物和退火温度可供参考。其中，产物长度为约数，不作为判别标准。表5近交系大鼠38个SNP标记检测位点序号SNPID序号SNPID序号SNPID1rs6475023714rsl0661048927rsl34532522rs10688823615rsl345311528rs641027873rs10645839616rsl0575376029rs1066474054rs10703834717rs817331130rs655455625rs10667225818rs6619019831rs1056945456rs10546630919rsl0650616132rsl34580247rs6592130520rsl0699696133rs81660898rs10679599321rs6572967734rs81581779rsl0689178822rs6597029335rsl345731610rs6580192023rs817094736rsl344859111rs816488024rs10709776337rs1345623112rs10500866525rs817325738rs814905313rs815254126rsl05187832/表6近交系大鼠38个SNP标记引物及退火温度NoprimersequenceProduct(bp)Tm()Chr1rs64750237CagtgtagctcaggcagatCcaggcaccatcaaggaa1595842rsl06888236TgtgaagaacacaggctctccTgccaaacaccatgtaatgaa20358163rs106458396GagaatgagaaggccacactCaccaaagagatagcgaagc26658154rsl07038347TagtcacctagtccttcagaTtacaagtcagtggcagaa3035835rsl06672258TctggctgtgatcggaactaCaacgaatgagcaagagaagac37858126rs105466309GCGAGCCCTCACGAATCCnGTGTTGCCTATGGATGA4685857rs65921305GctgtgcttggcataatatcCcaggtgattatagattgtgtc55558138rs106795993AacagcagccgcttcttagCagtgtcagatgtggtggtata70258119rs106891788GgtgccttcctgacaaagtGggtttccattgctatttgaca15658110rs65801920TggctcttggtcatcatctgtTctgttcttggtctgctgagt19658911rs8164880CgtctgtgttagtaggtgatgcCctctgatgctgttggtatgc261581412rs105008665CatctgcctgctacaactgttTCnGTATTGGTCAGCACTCTC35558213rs8152541CaggtggtgatggtgtaagcCctcatgcagtagcgagtaac430581814rs106610489CgctgtccaatctcctgaagCtagaagccattgtctggtcaa49958215rsl3453115CtgacttgacagaacggtaggAgagcagtgaaggtgaggag597581516rs105753760AgcagcatcacaatagcaaggCttccctaacacatccctacca788581417rs8173311CcaccgcatttgaccagtttGccctgtgtttgatggagtag359581918rs66190198GagttcctgagtgcttgattgtAtgggagttaaggcggtttct214581119rsl06506161GcagcaggacacaaggaagTcttaggagaccagaggagtg268581020rs106996961TgtgtctctgtagttggtgttgGgatatggtgcagtcgtcatt36658221rs65729677AacctcaattcaacagccagaaCcacgactcacctacattcac48058522rs65970293CtctggttggtctggttggttAgatgcggatgccattaagtct53958123rs8170947GacacagtcagcatgtaagccAgacagtcactcacaactcaga67958224rsl07097763TGTCGGGATCTTCTGGA19158225262728293031323334353637Cagtctcctcctgaatgtgaatrs8173257AgtgtgactgaccgaagaagAactcctgcctctacttaccta3315817rsl05187832GgctttcccttctccatacttgTtacctgtgactcctgcttgt300587rs13453252CctgtaatcaagatacgccattGctaggaccttaaccaccata3495820rs64102787GctgggctgtggttagactAgcatggctggcagttca406586rs106647405CcnCTTCTGGAACCTGCTCTGGaccctgtaattggcacctgtt469584rs65545562TgctgctctaacgatgagtacaGatgtaggcaccaggcttgt5855821rsl05694545CctcctatccgtgagtcctgagTtctgg