專利名稱:心腦血管疾病易感基因芯片檢測試劑盒的制作方法
技術領域:
本發(fā)明屬于臨床檢測的基因芯片技術領域,具體地說涉及一種心腦血管疾病易感基因芯 片檢測試劑盒。
背景技術:
與心腦血管疾病相關的易感基因比較多,通過査閱文獻,挑選出13個相關性較強的基因, 分別是Enos基因,ADD1基因,AP0B-2515基因,AT1R基因,APOB-3500基因,APOB-4181 基因,eNOS-894基因,F(xiàn)gB-148基因,APOE基因,GNB3基因,AGT基因,F(xiàn)VII基因, MTHFR基因。用以對心腦血管疾病易感基因進行相關研究。目前用于對心腦血管疾病易感基因研究的平行性沒有達到13個基因以上的,本試劑盒的 易感基因概括全面。另外對心腦血管疾病易感基因相關基因點突變的檢測方法主要有測序 法,這是最直接,最準確的方法;熒光定量PCR法,和單堿基延伸法。這些方法所需要的設 備儀器都價格昂貴,技術復雜,從經(jīng)濟上考慮不適于用作常規(guī)方法加以推廣。發(fā)明內(nèi)容為了對心腦血管疾病易感的相關基因進行平行檢測,對大量樣本進行同時分析,并考慮 經(jīng)濟推廣的可行性,本發(fā)明提供一種心腦血管疾病易感基因芯片檢測試劑盒,包括抽提液、 雜交緩沖液、洗液、擴增液、Taq酶、基因芯片;其特征在于,所含的擴增液含有擴增13個 含突變位點的基因,分別是Enos基因,ADD1基因,AP0B-2515基因,AT1R基因,APOB-3500 基因,APOB-4181基因,eNOS-894基因,F(xiàn)gB-148基因,APOE基因,GNB3基因,AGT 基因,F(xiàn)VII基因,MTHFR基因。所述的心腦血管疾病易感基因芯片檢測試劑盒,擴增液可以包含以下一組序列的引物- :Enos-l: CCAGATGCCCAGCTAG Enos-2: GACATTAGGGTATCCCTT ADD1-1: CACAGGTTACTCCTTTGCTA ADD1陽2: GGTTTCACGTGATCGTGT APOB-2515-l: AACCAAGGCCACAGTTGC APOB-2515-2: CTCTACCAATGCTTTCATA AT1R陽1: CATGTTCGAAACCTGTCCA AT1R-2: TCGAGTTTCTGACATTGTTC APOB-3500-1: AATGTCAGCTCTTGTTCAGG APOB-3500-2: CCAGCTTTAGGAAATCC APOB-4181 -1: GGCCATTAGGCAAATTGAT APOB-4181-2: CCTCCCTTATGAACATAGT eNOS-894-l: ATTCAGCACGGCTGGA eNOS-894-2: TGCAGGCCCTTCTTGA FgB-148-l: CGGAGCTTGTGTAGTTTCFgB-148-2: TGGAAGCTCCAAGAAACC APOE-h CGAGACCATGAAGGAGTT APOE-2: CTGCCCATCTCCTCCA GNB3-1: GATCCCTGACCCACTTG GNB3-2: TTACCCACACGCTCAGAC AGT-1: GTTGCTGCTGAGAAGATT AGT-2: CCATCTCCAAGGCCTG FVII-1: TCATGGTCCTCAACGT FVII-2: TTTCTCTCCACAGGCC MTHFR-1: CCTGAAGCACTTGAAGG MTHFR-2: GGGTGGAGGGAGCTTA所述的心腦血管疾病易感基因芯片檢測試劑盒,還可以包括擴增液B,擴增液B可以包 含如下一組序列的ASPE (Allele-Specific Primer Extension,即等位基因特特性引物延伸)引物Enos-丁 agctcttccctggcT Enos-G: agctcttccctggcC ADD1-G: aagcttccgaggaaG ADD1陽T: aagcttccgaggaaT APOB-2515-C: aaattccgagagacC APOB-2515-T: aattccgagagacT AT1R-A: ttcactaccaaatgagcA AT1R-C: ttcactaccaaatgagcC APOB-3500-G: atggaaaaatgaagtccG atggaaaaatgaagtccA ggtacgagttactcaaG ggtacgagttactcaaA eNOS-894國G: aggccccagatgaG eNOS陽894-T: aggccccagatgaT FgB-148-C: gtatgacaagtaaataagC FgB-148-T: gtatgacaagtaaataagT APOE-陽Pl: acatggaggacgtgT APOE-P2: gacatggaggacgtgC APOE- P3: gatgacctgcagaagC APOE-P4: gacctgcagaagT GNB3-C: ctgcggcatcacgtcC GNB3-T: ctgcggcatcacgtcT AGT-T: ctggctgctccctgaT AGT-C: ctggctgctccctgaC FVII-G: tgccacccactaccGAPOB-3500陽A APOB-4181-G APOB-4181-AFVII-A: tgccacccactaccA MTHFR-C: GGTGTCTGCGGGAGC MTHFR-T: GGTGTCTGCGGGAGT所述的心腦血管疾病易感基因芯片檢測試劑盒,所述的擴增液B中包含的ASPE引物還 可以是含有5'-端檢測定位序列的如下序列Enos-tagT: CTTTAATCTCAATCAATACAAATCagctc加cctggcTEnos-tagC: CTTTATCAATACATACTACAATCAagctcttccctggcCADDl-tagG: TACACTTTATCAAATCTTACAATaagcttccgaggaaGADD 1 -tagT: TAC ATTACC AATAATCTTC AAATCaagcttccgaggaaTAPOB-2515陽tagC: CAATTCAAATCACAATAATCAATCaaattccgagagacCAPOB-2515-tagT: TCAACAATCTTTTACAATCAAATCaattccgagagacTAT 1 R-tagA: CAATTCATTTACCAATTTACCAATttcactaccaaatgagcAATlR-tagC: AATCCTTTTACATTCATTACTTACttcactaccaaatgagcCAPOB-3500-tagG: TAATCTTCTATATCAACATCTTACatggaaaaatgaagtccGAPOB-3500-tagA: ATCATACATACATACAAATCTACAatggaaaaatgaagtccAAPOB-4181 -tagG: TACAAATCATCAATCACTTTAATggtacgagttactcaaGAPOB-4181 -tagA: CAATAAACTATACTTCTTCACTAAggtacgagttactcaaAeNOS-894國tagG: CTACTATACATCTTACTATACTTTaggccccagatgaGeNOS-894-tagT: ATACTTCATTCATTCATCAATTCAcaggccccagatgaTFgB-148-tagC: AATCAATCTTCATTCAAATCATCAgtatgacaagtaaataagCFgB-148-tagT: AATCCTTTCTTTAATCTCAAATCAgtatgacaagtaaataagTAPOE-tag-Pl: TTACTCAAAATCTACACTTTTTCAacatggaggacgtgTAPOE-tag-P2: CTACAAACAAACAAACATTATCAAgacatggaggacgtgCAPOE-tag-P3: TTCACTTTTCAATCAACTTTAATCgatgacctgcagaagCAPOE-tag-P4: TCAATTACTTCACTTTAATCCTTTgacctgcagaagTGNB3-tagC: CAATTCATTTCATTCACAATCAATctgcggcatcacgtcCGNB3-tagT: CAATATACCAATATCATCATTTACctgcggcatcacgtcTAGT-tagT: TCAATCATCTTTATACTTCACAATctggctgctccctgaTAGT-tagC: CTTTTTCAATCACTTTCAATTCATctggctgctccctgaCFVII-tagG: CAATTTACTCATATACATCACTTTtgccacccactaccGFVII-tagA: CTACTAATTCATTAACATTACTACtgccacccactaccAMTHFR-tagC: CTTTAATCCTTTATCACTTTATCAGGTGTCTGCGGGAGCMTHFR-tagT: TC AATC AATTACTTACTC AA ATACGGTGTCTGCGGGAGT所述的心腦血管疾病易感基因芯片檢測試劑盒,擴增液B含有生物素標記的dCTP。所述的心腦血管疾病易感基因芯片檢測試劑盒,所含的基因芯片上的探針序列可以是以下序列POS-1: GATTTGTATTGATTGAGATTAAAG POS陽2: TGATTGTAGTATGTATTGATAAAG POS陽3: GATTGTAAGATTTGATAAAGTGTAPOS-4: GATTTGAAGATTATTGGTAATGTA POS-5: GATTGATTATTGTGATTTGAATTG POS-6: GATTTGATTGTAAAAGATTGTTGA POS-7: ATTGGTAAATTGGTAAATGAATTG POS-8: GTAAGTAATGAATGTAAAAGGATT POS-9: GTAAGATGTTGATATAGAAGATTA POS-10: TGTAGATTTGTATGTATGTATGAT POS-11: GATTAAAGTGATTGATGATTTGTA POS墨12: TTAGTGAAGAAGTATAGTTTATTG POS-13: AAAGTATAGTAAGATGTATAGTAG POS-14: TGAATTGATGAATGAATGAAGTAT POS-15: TGATGATTTGAATGAAGATTGATT POS-16: TGATAAAGTGATAAAGGATTAAAG POS-17: GTATTTGAGTAAGTAATTGATTGA POS-18: TGATTTGAGATTAAAGAAAGGATT POS-19: TGAAAAAGTGTAGATTTTGAGTAA POS-20: TTGATAATGTTTGTTTGTTTGTAG POS-21: GATTAAAGTTGATTGAAAAGTGAA POS-22: AAAGGATTAAAGTGAAGTAATTGA POS-23: ATTGATTGTGAATGAAATGAATTG POS-24: GTAAATGATGATATTGGTATATTG POS-25: ATTGTGAAGTATAAAGATGATTGA POS-26: ATGAATTGAAAGTGATTGAAAAAG POS-27: AAAGTGATGTATATGAGTAAATTG POS-28: GTAGTAATGTTAATGAATTAGTAG該試劑盒通過將與心腦血管疾病相關的13個易感基因的檢測結合在一起,達到對相關基 因進行平行檢測的目的。另外通過將ASPE法與化學發(fā)光法相結合,檢測單位點突變,優(yōu)點在 于(1)結果準確,(2)通量大,(3)背景低,(4)操作簡便,(5)無需大型儀器。
圖1為芯片檢測試劑盒應用流程2為試劑盒中所含心腦血管疾病易感基因檢測芯片示意圖具體實施方式
下面結合附圖和舉例說明本發(fā)明目的的實施。實施例子1. 樣本處理用口腔粘膜脫落細胞作為樣本來源,進行基因組DNA抽提。抽提使用博大泰克的細胞 基因組抽提試劑盒。2. 芯片制備,將文獻中已有標記定位探針在超平玻片上點樣,探針序列如下POS-l: GATTTGTATTGATTGAGATTAAAG POS-2: TGATTGTAGTATGTATTGATAAAG POS-3: GATTGTAAGATTTGATAAAGTGTA POS-4: GATTTGAAGATTATTGGTAATGTA POS-5: GATTGATTATTGTGATTTGAATTG POS-6: GATTTGATTGTAAAAGATTGTTGA POS陽7: ATTGGTAAATTGGTAAATGAATTG POS-8: GTAAGTAATGAATGTAAAAGGATT POS-9: GTAAGATGTTGATATAGAAGATTAPOS-10:TGTAGATTTGTATGTATGTATGATPOS-ll:GATTAAAGTGATTGATGATTTGTAPOS-12:TTAGTGAAGAAGTATAGTTTATTGPOS-13:AAAGTATAGTAAGATGTATAGTAGPOS-14:TGAATTGATGAATGAATGAAGTATPOS-15:TGATGATTTGAATGAAGATTGATTPOS-16:TGATAAAGTGATAAAGGATTAAAGPOS-17:GTATTTGAGTAAGTAATTGATTGAPOS陽18:TGATTTGAGATTAAAGAAAGGATTPOS-19:TGAAAAAGTGTAGATTTTGAGTAAPOS陽20:TTGATAATGTTTGTTTGTTTGTAGPOS-21:GATTAAAGTTGATTGAAAAGTGAAPOS-22:AAAGGATTAAAGTGAAGTAATTGAPOS-23:ATTGATTGTGAATGAAATGAATTGPOS陽24:GTAAATGATGATATTGGTATATTGPOS-25:ATTGTGAAGTATAAAGATGATTGAPOS-26:ATGAATTGAAAGTGATTGAAAAAGPOS-27:AAAGTGATGTATATGAGTAAATTGPOS-28:GTAGTAATGTTAATGAATTAGTAG3.引物設計(1)設計以下引物序列組A,用以擴增與心腦血管疾病易感性相關的,含突變位點的13個基因片斷Enos-l: CCAGATGCCCAGCTAG Enos-2: GACATTAGGGTATCCCTT ADD 1 -1: CACAGGTTACTCCTTTGCTA ADD1陽2: GGTTTCACGTGATCGTGTAPOB-2515-1: AACCAAGGCCACAGTTGC APOB-2515-2: CTCTACCAATGCTTTCATA AT1R-1: CATGTTCGAAACCTGTCCA AT1R-2: TCGAGTTTCTGACATTGTTC APOB-3500-1: AATGTCAGCTCTTGTTCAGG APOB-3500-2: CCAGCTTTAGGAAATCC APOB-4181-1: GGCCATTAGGCAAATTGAT APOB-4181-2: CCTCCCTTATGAACATAGT eNOS-894-l: ATTCAGCACGGCTGGA eNOS-894-2: TGCAGGCCCTTCTTGA FgB-148-1: CGGAGCTTGTGTAGTTTC FgB-148-2: TGGAAGCTCCAAGAAACC APOE-l: CGAGACCATGAAGGAGTT APOE-2: CTGCCCATCTCCTCCA GNB3-1: GATCCCTGACCCACTTG GNB3-2: TTACCCACACGCTCAGAC AGT-1: GTTGCTGCTGAGAAGATT AGT-2: CCATCTCCAAGGCCTG FVII-1: TCATGGTCCTCAACGT FVII-2: TTTCTCTCCACAGGCC MTHFR-1: CCTGAAGCACTTGAAGG MTHFR陽2: GGGTGGAGGGAGCTTA (2)設計以下ASPE引物序列組B,用以擴增含突變位點的序列,同時引進用于基因檢測定 位的標記序列,以便與芯片上相應的定位探針雜交,從而獲得檢測結果。 Enos誦tagT: CTTTAATCTCAATCAATACAAATCagctcttccctggcT Enos-tagC: CTTTATCAATACATACTACAATCAagctcttccctggcC ADD 1 -tagG: TACACTTTATCAAATCTTACAATaagcttccgaggaaG ADDl-tagT: TACATTACCAATAATCTTCAAATCaagcttccgaggaaT APOB-2515-tagC: CAATTCAAATCACAATAATCAATCaaattccgagagacC APOB-2515-tagT: TCAACAATCTTTTACAATCAAATCaattccgagagacT ATlR-tagA: CAATTCATTTACCAATTTACCAATttcactaccaaatgagcA ATlR-tagC: AATCCTTTTACATTCATTACTTACttcactaccaaatgagcC APOB-3500國tagG: TAATCTTCTATATCAACATCTTACatggaaaaatgaagtccG APOB-3500-tagA: ATCATACATACATACAAATCTACAatggaaaaatgaagtccA APOB陽4181 -tagG: TACAAATCATCAATCACTTTAATggtacgagttactcaaG APOB-4181隱tagA: CAATAAACTATACTTCTTCACTAAggtacgagttactcaaA eNOS-894-tagG: CTACTATACATCTTACTATACTTTaggccccagatgaG eNOS-894-tagT: ATACTTCATTCATTCATCAATTCAcaggccccagatgaT FgB-148-tagC: AATCAATCTTCATTCAAATCATCAgtatgacaagtaaataagCFgB-148-tagT: AATCCTTTCTTTAATCTCAAATCAgtatgacaagtaaataagT APOE-tag-Pl: TTACTCAAAATCTACACTTTTTCAacatggaggacgtgT APOE-tag-P2: CTACAAACAAACAAACATTATCAAgacatggaggacgtgC APOE-tag-P3: TTCACTTTTCAATCAACTTTAATCgatgacctgcagaagC APOE-tag-P4: TCAATTACTTCACTTTAATCCTTTgacctgcagaagT GNB3-tagC: CAATTCATTTCATTCACAATCAATctgcggcatcacgtcC GNB3-tagT: CAATATACCAATATCATCATTTACctgcggcatcacgtcT AGT-tagT: TCAATCATCTTTATACTTCACAATctggctgctccctgaT AGT-tagC: CTTTTTCAATCACTTTCAATTCATctggctgctccctgaC FVII-tagG: CAATTTACTCATATACATCACTTTtgccacccactaccG FVII-tagA: CTACTAATTCATTAACATTACTACtgccacccactaccA MTHFR國tagC: CTTTAATCCTTTATCACTTTATCAGGTGTCTGCGGGAGC MTHFR-tagT: TCAATCAATTACTTACTCAAATACGGTGTCTGCGGGAGT4. PCR擴增待測片斷并引入生物素標記。用引物序列組A擴增含待測突變位點的序列,用ASPE 引物序列組B擴增含突變位點的序列,擴增時加入含生物素標記的dCTP,以此引入對樣本產(chǎn) 物檢測所需的生物素標記。5. 雜交檢測按以下步驟對得到的相應樣本產(chǎn)物進行雜交檢測(1) 將擴增得到的標記產(chǎn)物與雜交液(5XSSC,0.2%SDS)以1:6的比例混合,加入反應 區(qū)域,50ul。(2) 42'C保溫保濕反應60分鐘。(3) 用洗液(2XSSC,0. 1%SDS)洗滌4-6次,每次2分鐘,振動。(4) 加入HRP標記的親和素反應液,50ul。(5) 在42。C反應15-20min。(6) 用洗液(2XSSC,0. 1%SDS)洗滌4-6次,每次2分鐘,晃動。(7) 加檢測液20ul/孔,使檢測液均勻分布于反應區(qū)域。(8) CCD檢測,視發(fā)光信號強度曝光30-60秒。6. 結果判定依據(jù)不同指標的CutOff值,參考標準品梯度曲線,判定樣本的陰陽性。 與此同時,用常規(guī)方法對測試的樣本進行常規(guī)檢測。結果表明,用本發(fā)明的試劑盒得出 的測試結果與常規(guī)方法相同。序列表Enos-l: CCAGATGCCCAGCTAG Enos-2: GACATTAGGGTATCCCTT ADD 1 -1: CACAGGTTACTCCTTTGCTA ADD 1-2: GGTTTCACGTGATCGTGT APOB-2515-1: AACCAAGGCCACAGTTGC APOB-2515-2: CTCTACCAATGCTTTCATA AT1R-1: CATGTTCGAAACCTGTCCA AT1R-2: TCGAGTTTCTGACATTGTTC APOB-3500-1: AATGTCAGCTCTTGTTCAGG APOB-3500-2: CCAGCTTTAGGAAATCC APOB-4181-1: GGCCATTAGGCAAATTGAT APOB-4181-2: CCTCCCTTATGAACATAGT eNOS-894-1: ATTCAGCACGGCTGGA eNOS-894-2: TGCAGGCCCTTCTTGA FgB-148-1: CGGAGCTTGTGTAGTTTC FgB-148-2: TGGAAGCTCCAAGAAACC APOE-1: CGAGACCATGAAGGAGTT APOE-2: CTGCCCATCTCCTCCA GNB3-1: GATCCCTGACCCACTTG GNB3-2: TTACCCACACGCTCAGAC AGT-1: GTTGCTGCTGAGAAGATT AGT-2: CCATCTCCAAGGCCTG FVII-1: TCATGGTCCTCAACGT FVII-2: TTTCTCTCCACAGGCC MTHFR-1: CCTGAAGCACTTGAAGG MTHFR-2: GGGTGGAGGGAGCTTAEnos-T: agctcttccctggcT Enos陽G: agctcttccctggcC ADD1-G: aagettccgaggaaG ADD 1 -T: aagcttccgaggaaT APOB-2515-C: aaattccgagagacC APOB-2515-T: aattccgagagacT AT1R-A: ttcactaccaaatgagcA AT1R-C: ttcactaccaaatgagcC APOB-3500-G: atggaaaaatgaagtccG APOB-3500-A: atggaaaaatgaagtccA APOB-4181-G: ggtacgagttactcaaG APOB-4181 -A: ggtacgagttactcaaA eNOS-894-G: aggccccagatgaG eNOS-894-T: aggccccagatgaT FgB-148-C: gtatgacaagtaaataagC FgB-148-T: gtatgacaagtaaataagT APOE—PI: acatggaggacgtgT APOE-P2: gacatggaggacgtgC APOE- P3: gatgacctgcagaagC APOE-P4: gacctgcagaagT GNB3 -C: ctgcggcatcacgtcC GNB3-T: ctgcggcatcacgtcT AGT隱T: ctggctgctccctgaT AGT-C: ctggctgctccctgaC FVII-G: tgccacccactaccG FVII-A: tgccacccactaccA MTHFR-C: GGTGTCTGCGGGAGC MTHFR-T: GGTGTCTGCGGGAGTEnos-tagT: CTTTA ATCTC AATC AATAC AAATCagctcttccctggcT Enos-tagC: CTTTATC AATAC ATACTACAATCAagctcttccctggcC ADD 1 -tagG: TAC ACTTTATC AAATCTTAC AATaagcttccgaggaaG ADD 1 -tagT: TAC ATTACC A ATAATCTTC AAATCaagcttccgaggaaT APOB-2515-tagC C AATTCAAATCACAATAATC AATCaaattccgagagacC APOB-2515-tagT: TCAAC AATCTTTTAC AATCAAATCaattccgagagacT AT 1 R-tag A: C AATTC ATTTACC AATTTACC AATttcactaccaaatgagc A ATlRtagC: AATCCTTTTACATTCATTACTTACttcactaccaaatgagcC APOB-3 500-tagG: TAATCTTCTATATC AAC ATCTTACatggaaaaatgaagtccG APOB3500-tagA: ATCATACATACATACAAATCTAC AatggaaaaatgaagtccA APOB-4181 -tagG: TAC A AATC ATC AATC ACTTTA ATggtacgagttactcaaG APOB-4181 -tagA: CAATAAACTATACTTCTTCACTAAggtacgagttactcaaA eNOS-894-tagG: CTACTATAC ATCTTACTATACTTTaggccccagatgaG eNOS-894-tagT: ATACTTC ATTC ATTC ATCAATTCAcaggccccagatgaT FgB-148-tagC: AATC A ATCTTC ATTC AA ATC ATC AgtatgacaagtaaataagC FgB-148-tagT: AATCCTTTCTTTAATCTCAAATC AgtatgacaagtaaataagT APOE-tag-Pl: TTACTCAAAATCTACACTTTTTCAacatggaggacgtgT APOEtag-P2: CTACAAAC AAAC AAAC ATTATC AAgacatggaggacgtgC APOE-tag-P3: TTCACTTTTCAATCAACTTTAATCgatgacctgcagaagC APOE-tag-P4: TC AATTACTTC ACTTTAATCCTTTgacctgcagaagT GNB3-tagC: CAATTCATTTCATTCAC AATCAATctgcggcatcacgtcC GNB3-tagT: CAATATACCAATATCATCATTTACctgcggcateacgtcT AGT-tagT: TC AATC ATCTTTATACTTC AC A ATctggctgctccctgaT AGT-tagC: CTTTTTC AATC ACTTTC A ATTC ATctggctgctccctgaC FVII-tagG: CAATTTACTCATATACATCACTTTtgccacccactaccG FVII-tagA: CTACTAATTC ATTAAC ATTACTACtgccacccactacc A MTHFR-tagC: CTTTAATCCTTTATCACTTTATCAGGTGTCTGCGGGAGC MTHFR-tagT: TC AATC AATTACTTACTC A AATACGGTGTCTGCGGGAGTPOS-1: GATTTGTATTGATTGAGATTAAAG POS-2: TGATTGTAGTATGTATTGATAAAG POS-3: GATTGTAAGATTTGATAAAGTGTA POS-4: GATTTGAAGATTATTGGTAATGTA POS-5: GATTGATTATTGTGATTTGAATTG POS-6: GATTTGATTGTAAAAGATTGTTGA POS-7: ATTGGTAAATTGGTAAATGAATTG POS-8: GTAAGTAATGAATGTAAAAGGATT POS-9: GTAAGATGTTGATATAGAAGATTA POS-10: TGTAGATTTGTATGTATGTATGAT POS-11: GATTAAAGTGATTGATGATTTGTA POS-12: TTAGTGAAGAAGTATAGTTTATTG POS-13: AAAGTATAGTAAGATGTATAGTAG POS-14: TGAATTGATGAATGAATGAAGTAT POS-15: TGATGATTTGAATGAAGATTGATT POS-16: TGATAAAGTGATAAAGGATTAAAG POS-17: GTATTTGAGTAAGTAATTGATTGA POS-18: TGATTTGAGATTAAAGAAAGGATT POS-19: TGAAAAAGTGTAGATTTTGAGTAA POS-20: TTGATAATGTTTGTTTGTTTGTAG POS-21: GATTAAAGTTGATTGAAAAGTGAA POS-22: AAAGGATTAAAGTGAAGTAATTGA POS-23: ATTGATTGTGAATGAAATGAATTG POS-24: GTAAATGATGATATTGGTATATTG POS-25: ATTGTGAAGTATAAAGATGATTGA POS-26: ATGAATTGAAAGTGATTGAAAAAG POS-27: AAAGTGATGTATATGAGTAAATTG POS-28: GTAGTAATGTTAATGAATTAGTAG
權利要求
1.一種心腦血管疾病易感基因芯片檢測試劑盒,包括抽提液、雜交緩沖液、洗液、擴增液、Taq酶、基因芯片;其特征在于,所含的擴增液含有擴增13個含突變位點的基因,分別是Enos基因,ADD1基因,APOB-2515基因,AT1R基因,APOB-3500基因,APOB-4181基因,eNOS-894基因,F(xiàn)gB-148基因,APOE基因,GNB3基因,AGT基因,F(xiàn)VII基因,MTHFR基因。
2. 根據(jù)權利要求1所述的心腦血管疾病易感基因芯片檢測試劑盒,其特征在于,所述的擴 增液可以包含以下一組序列的引物Enos-l: CCAGATGCCCAGCTAG Enos-2: GACATTAGGGTATCCCTT ADD 1 -1: CACAGGTTACTCCTTTGCTA ADD 1-2: GGTTTCACGTGATCGTGT APOB-2515-1: AACCAAGGCCACAGTTGC APOB-2515-2: CTCTACCAATGCTTTCATA AT1R-1: CATGTTCGAAACCTGTCCA AT1R-2: TCGAGTTTCTGACATTGTTC APOB-3500-1: AATGTCAGCTCTTGTTCAGG APOB-3500-2: CCAGCTTTAGGAAATCC APOB-4181-1: GGCCATTAGGCAAATTGAT APOB-4181-2: CCTCCCTTATGAACATAGT eNOS-894-1: ATTCAGCACGGCTGGA eNOS-894-2: TGCAGGCCCTTCTTGA FgB-148-1: CGGAGCTTGTGTAGTTTC FgB墨148-2: TGGAAGCTCCAAGAAACC APOE-1: CGAGACCATGAAGGAGTT APOE-2: CTGCCCATCTCCTCCA GNB3-1: GATCCCTGACCCACTTG GNB3陽2: TTACCCACACGCTCAGAC AGT-1: GTTGCTGCTGAGAAGATT AGT-2: CCATCTCCAAGGCCTG FVII -h TCATGGTCCTCAACGT FVII-2: TTTCTCTCCACAGGCC MTHFR陽l: CCTGAAGCACTTGAAGG MTHFR-2: GGGTGGAGGGAGCTTA
3. 根據(jù)權利要求1或2所述的心腦血管疾病易感基因芯片檢測試劑盒,其特征在于,還可 以包括擴增液B,擴增液B可以包含如下一組序列的ASPE (Allele-Specific Primer Extension, 即等位基因特特性引物延伸)引物Enos-T: agctcttccctggcT Enos-C: agctcttccctggcC ADD 1 -G: aagcttccgaggaaGADD1-T: aagcttccgaggaaTAPOB-2515-C: aaattccgagagacCAPOB-2515-T: aattccgagagacTAT 1R-A: ttcactaccaaatgagcAAT1R-C: ttcactaccaaatgagcCAPOB-3500-G: atggaaaaatgaagtccGAPOB-3500-A: atggaaaaatgaagtccAAPOB-4181-G: ggtacgagttactcaaGAPOB隱4181 - A: ggtacgagttactcaaAeNOS-894-G: aggccccagatgaGeNOS-894-T: aggccccagatgaTFgB-148-C: gtatgacaagtaaataagCFgB-148陽T: gtatgacaagtaaataagTAPOE—Ph acatggaggacgtgTAPOE-P2: gacatggaggacgtgCAPOE- P3: gatgacctgcagaagCAPOE-P4: gacctgcagaagTGNB3 -C: ctgcggcatcacgtcCGNB3-T: ctgcggcatcacgtcTAGT-T: ctggctgctccctgaTAGT-C: ctggctgctccctgaCFVII-G: tgccacccactaccGFVII-A: tgccacccactaccAMTHFR-C: GGTGTCTGCGGGAGCMTHFR陽T: GGTGTCTGCGGGAGT
4.根據(jù)權利要求3所述的心腦血管疾病易感基因芯片檢測試劑盒,其特征在于,所述的擴 增液B中包含的ASPE引物還可以是含有5'-端檢測定位序列的如下序列Enos陽tagT: CTTTAATCTCAATCAATACAAATCagctc加cctggcTEnos-tagC: CTTTATCAATACATACTACAATCAagctcttccctggcCADD 1 -tagG: TACACTTTATCAAATCTTACAATaagcttccgaggaaGADDl-tagT: TACATTACCAATAATCTTCAAATCaagcttccgaggaaTAPOB-2515-tagC: CAATTCAAATCACAATAATCAATCaaattccgagagacCAPOB-2515-tagT: TCAACAATCTTTTACAATCAAATCaattccgagagacTATlR-tagA: CAATTCATTTACCAATTTACCAATttcactaccaaatgagcAATlR-tagC: AATCCTTTTACATTCATTACTTACttcactaccaaatgagcCAPOB-3500-tagG: TAATCTTCTATATCAACATCTTACatggaaaaatgaagtccGAPOB-3500-tagA: ATCATACATACATACAAATCTACAatggaaaaatgaagtccAAPOB-4181 -tagG: TACAAATCATCAATCACTTTAATggtacgagttactcaaGAPOB-4181 -tagA: CAATAAACTATACTTCTTCACTAAggtacgagttactcaaAeNOS-894-tagG: CTACTATACATCTTACTATACTTTaggccccagatgaG eNOS-894-tagT: ATACTTCATTCATTCATCAATTCAcaggccccagatgaT FgB-148-tagC: AATCAATCTTCATTCAAATCATCAgtatgacaagtaaataagC FgB-148-tagT: AATCCTTTCTTTAATCTCAAATCAgtatgacaagtaaataagT APOE-tag-Pl: TTACTCAAAATCTACACTTTTTCAacatggaggacgtgT APOE-tag-P2: CTACAAACAAACAAACATTATCAAgacatggaggacgtgC APOE-tag-P3: TTCACTTTTCAATCAACTTTAATCgatgacctgcagaagC APOE-tag-P4: TCAATTACTTCACTTTAATCCTTTgacctgcagaagT GNB3-tagC: CAATTC ATTTC ATTCAC AATC AATctgcggcatcacgtcC GNB3-tagT: C AATATACC AATATCATCATTTACctgcggcatcacgtcT AGT-tagT: TCAATCATCTTTATACTTCACAATctggctgctecctgaT AGT-tagC: CTTTTTCAATCACTTTCAATTCATctggctgctccctgaC FVII-tagG: CAATTTACTCATATACATCACTTTtgccacccactaccG FVII-tagA: CTACTAATTCATTAACATTACTACtgccacccactaccA MTHFR-tagC: CTTTAATCCTTTATCACTTTATCAGGTGTCTGCGGGAGC MTHFR陽tagT: TC A ATC AATTACTTACTC AAATACGGTGTCTGCGGG AGT
5. 根據(jù)權利要求3或4所述的心腦血管疾病易感基因芯片檢測試劑盒,其特征在于,所述 的擴增液B含有生物素標記的dCTP。
6. 根據(jù)權利要求4所述的心腦血管疾病易感基因芯片檢測試劑盒,其特征在于,所含的基 因芯片上的探針序列可以是以下序列POS-1: GATTTGTATTGATTGAGATTAAAG POS-2: TGATTGTAGTATGTATTGATAAAG POS-3: GATTGTAAGATTTGATAAAGTGTA POS-4: GATTTGAAGATTATTGGTAATGTA POS-5: GATTGATTATTGTGATTTGAATTG POS-6: GATTTGATTGTAAAAGATTGTTGA POS-7: ATTGGTAAATTGGTAAATGAATTG POS-8: GTAAGTAATGAATGTAAAAGGATT POS-9: GTAAGATGTTGATATAGAAGATTA POS-10: TGTAGATTTGTATGTATGTATGAT POS-11: GATTAAAGTGATTGATGATTTGTA POS-12: TTAGTGAAGAAGTATAGTTTATTG POS-13: AAAGTATAGTAAGATGTATAGTAG POS-14: TGAATTGATGAATGAATGAAGTAT POS-15: TGATGATTTGAATGAAGATTGATT POS-16: TGATAAAGTGATAAAGGATTAAAG POS-17: GTATTTGAGTAAGTAATTGATTGA POS-18: TGATTTGAGATTAAAGAAAGGATT POS-19: TGAAAAAGTGTAGATTTTGAGTAAPOS-20TTGATAATGTTTGTTTGTTTGTAGPOS-21GATTAAAGTTGATTGAAAAGTGAAPOS-22AAAGGATTAAAGTGAAGTAATTGAPOS-23ATTGATTGTGAATGAAATGAATTGPOS-24GTAAATGATGATATTGGTATATTGPOS-25ATTGTGAAGTATAAAGATGATTGAPOS-26ATGAATTGAAAGTGATTGAAAAAGPOS-27AAAGTGATGTATATGAGTAAATTGPOS-28GTAGTAATGTTAATGAATTAGTAG 。
全文摘要
本發(fā)明提供一種心腦血管疾病易感基因芯片檢測試劑盒,屬于臨床檢測的基因芯片技術領域,可用于平行經(jīng)濟地檢測與心腦血管疾病易感性相關的13個基因。該試劑盒包括抽提液、雜交緩沖液、洗液、擴增液、Taq酶、基因芯片,所述的擴增液含有擴增含突變位點的13個基因的引物,13個基因分別是Enos基因,ADD1基因,APOB-2515基因,AT1R基因,APOB-3500基因,APOB-4181基因,eNOS-894基因,F(xiàn)gB-148基因,APOE基因,GNB3基因,AGT基因,F(xiàn)VII基因,MTHFR基因。與心腦血管疾病易感性相關的基因很多,本發(fā)明可用于平行檢測相關的13個基因。
文檔編號C12Q1/68GK101333561SQ20071004322
公開日2008年12月31日 申請日期2007年6月29日 優(yōu)先權日2007年6月29日
發(fā)明者杜喜玲, 汪寧梅, 穆海東 申請人:上海裕隆生物科技有限公司