三、发明内容
针对上述现有技术存在的缺陷或不足,本发明的目的在于,提供一组用于检测血友病患者的FⅧ/FⅨ基因突变的引物,并利用引物进行血友病病因学分类。
为了实现上述任务,本发明采取如下的技术解决方案:
一组FⅧ/FⅨ基因突变引物在制备血友病病因学分类试剂中的用途,其特征在于,所述的引物为8对F9引物,39对、22倒位3条、1倒位4条F8引物,其中:
1.8对F9引物的序列如下:
HB-1F:CCCATTCTCTTCACTTGTCC;
HB-1R:CCTAGCTAACAAAGAACAGT;
HB-2+3F:AGAGATGTAAAATTTTCATGATGTT;
HB-2+3R:GCAGAAAAAACCCACATAAT;
HB-4F:CTACAGGGGAGGACCGGGCATTCTA;
HB-4R:AGTTTCAACTTGTTTCAGAGGGAA;
HB-5F:CATGAGTCAGTAGTTCCATGTACTTT;
HB-5R:TGTAGGTTTGTTAAAATGCTGAAGTT;
HB-6F:TTTAAATACTGATGGGCCTG;
HB-6R:GTTAGTGCTGAAACTTGCCT;
HB-7F:TAGGTCAGTGGTCCCAAGTAGTC;
HB-7R:GTCCCCCACTATCTCCTTGA;
HB-8-1F:TAAGAATGAGATCTTTAACA;
HB-8-1R:CTAAGGTAGTGAAGAACTAA;
HB-8-2F:GAAGAGTCTTCCACAAAGGG;
HB-8-2R:AAGATGGGAAAGTGATTAGTTA;
2.39对F8引物的序列如下:
HA-1F:gtagcgcgacggccagtAGCATCACAACCATCCTAAC;
HA-1R:cagggcgcagcgatgacGCACATCCAGTGGGTAAAGT;
HA-2F:TGGAAGCATTACTTCCAGCT;
HA-2R:AACTGAAACCTCAAGATTGG;
HA-3F:TGCTTCTCCACTGTGACCT;
HA-3R:ATCTAGTAAATGTAAGAAATACA;
HA-4F:AGCCCACAGATTTGAAGAGG;
HA-4R:ACGTTGCTGTGAGCCGAGAT;
HA-5F:CTTACTGTCAAGTAACTGATG;
HA-5R:CTTCATTCCTGAACAGTAATG;
HA-6F:TCCCACTTATTGTCATGGAC;
HA-6R:TACAGAACTGGTGCTGAA;
HA-7F:gtagcgcgacggccagtATAATGTCCCCTTCAGCAAC;
HA-7R:cagggcgcagcgatgacAGCCCAATGTTCTTGATAAA;
HA-8F:CCATATAGCCTGCAGAACAT;
HA-8R:CTGATGCTCAGCTATGTTAG;
HA-9F:gtagcgcgacggccagtCGAGTTTAGTGGGTGACATT;
HA-9R:cagggcgcagcgatgacTTTAGAGTTGGATTTGAGCC;
HA-10F:CTAGCCTCAAATTACTATAATG;
HA-10R:ACTTTAGACTGGAGCTTGAG;
HA-11F:gtagcgcgacggccagtAAGGGGACATACACTGAGAA;
HA-11R:cagggcgcagcgatgacGACACTTTCACAGTCAACCG;
HA-12F:TGCCATCGCTTTCATCATAG;
HA-12R:CATTCATTATTATCTGGACATCAC;
HA-13F:TTCCTGGGAATAAGATAATGG;
HA-13R:AGCATACGAATGGCTAGTGA;
HA-14aF:ACAGGCATAGTACAACAGCA;
HA-14aR:CTTGGCTATTCATTAAACCTG;
HA-14bF:TCCATCAGACAATTTGGCAG;
HA-14bR:CTACATTTTGCCTAGTGCTC;
HA-14CF:gtagcgcgacggccagtCACTGTATGTATCTGAGGCA;
HA-14CR:cagggcgcagcgatgacAGACAAACAAAACTTCCAAT;
HA-14dF:ATCTTCCAGCAGCATCTTAT;
HA-14dR:CCTCTACCCTCTTGTAAACC;
HA-14eF:gtagcgcgacggccagtGGATGACACCTCAACCCAGT;
HA-14eR:cagggcgcagcgatgacCCTTCCACGAGATCCAGATG;(https://www.daowen.com)
HA-14fF:gtagcgcgacggccagtTOCCTAOGGAAACTAGCAATG;
HA-14fR:cagggcgcagcgatgacTCACAAGAGCAGAGCAAAGG;
HA-15F:gtagcgcgacggccagtATTCCACTGTCCTTAACTCACCA;
HA-15R:cagggcgcagcgatgacACAAAACCCCAGCTTTCATG;
Ha-15F:gtagcgcgacggccagtAAAAATTTCCAAAAGTGGGA;
Ha-15R:cagggcgcagcgatgacTGGCAAGAGTATTTCAAGGA;
HA-16F:gtagcgcgacggccagtGAACCAGAGTGGATTTCTCA;
HA-16R:cagggcgcagcgatgacGAACCAGAGTGGATTTCTCA;
HA-17F:TGTCATTACTGACTGGAATCT;
HA-17R:CACTCCCACAGATATACTCT;
HA-18F:gtagcgcgacggccagtCCCAGTGCCTAGACCATTTAA;
HA-18R:cagggcgcagcgatgacTTGATCCAGGGACTTGAACAT;
HA-19F:CGCATAAACCAATGTATCTCATGCTC;
HA-19R:TCCTGACACAAGCAACCATTCC;
HA-20F:GACGTTCTCCCATTTTCATTG;
HA-20R:GGATTCATTATCTGAGATTCTCCACCAG;
HA-21F:CAGCTTAGATTAACCTTTCTC;
HA-21R:GAGTGAATGTGATACATTTCC;
HA-22F:TCAGGAGGTAGCACATACAT;
HA-22R:GTCCAATATCTGAAATCTGC;
HA-23F:GTCTTATGTAGATGTTGGATG;
HA-23R:AGTCTCAGGATAACTAGAACA;
HA-24F:GCTCAGTATAACTGAGGCTG;
HA-24R:CCCATAACCAAACTTCCTTGACAC;
HA-25F:AGTGCTGTGGTATGGTTAAG;
HA-25R:TTGCTCTGAAAATTTGGTCATA;
HA-26AF:gtagcgcgacggccagtCTCTGAGAGGCCTAACTTTT;
HA-26AR:cagggcgcagcgatgacATCCTGGACTACTGGAAACA;
HA-26bF:gtagcgcgacggccagtGGAGAAACCTGCATGAAAGC;
HA-26bR:cagggcgcagcgatgacTTGGCCATCACAAATTTCAA;
HA-26cF:gtagcgcgacggccagtTGCAAATGTGCATTTTTCTGA;
HA-26cR:cagggcgcagcgatgacCCTCCAGCCCCCTTTACTAT;
HA-26dF:gtagcgcgacggccagtCCACCCCCATAAGATTGTGA;
HA-26dR:cagggcgcagcgatgacCTGAAGAAACCAGCAGGAAAA;
HA-26EF:gtagcgcgacggccagtTCTTGCTATTCAGTGCCCCTA;
HA-26ER:cagggcgcagcgatgacGCTTGACCCTTATCTGACCTCTT;
HA-P1F:gtagcgcgacggccagtACAGAAAGAAGCAGGTGGAG;
HA-P1R:cagggcgcagcgatgacGAAGAGGGTTGGAGTAGGC;
HA-P2F:gtagcgcgacggccagtGAGGGATGGAGAAGTCAAAGTG;
HA-P2R:cagggcgcagcgatgacGAAATCTGGGGGAAAGGTGT;
HA-P3F:gtagcgcgacggccagtACCTCAACACTTGCTATTTC;
HA-P3R:cagggcgcagcgatgacAGTGTTTATCAAAGGGGCT;
3.22倒位3条F8引物的序列如下:
HA-P:TGCCTGTCCATTACACTGATGACATTATGCTGA;
HA-Q:TACAACCATTCTGCCTTTCACTTTCAGTGCAATA;
HA-B:CCAAACTATAACCAGCACCTTGAACTTCCCCTCTCA;
4.1倒位4条F8引物的序列如下:
HA-9F:GTTGTTGGGAATGGTTACGG;
HA-9cR:CTAGCTTGAGCTCCCTGTGG;
HA-intlh-2F:GGCAGGGATCTTGTTGGTAAA;
HA-intlh-2R:TGGGTGATATAAGCTGCTGAGCTA。
经申请人的试验证明,上述用于检测血友病患者的FⅧ/FⅨ基因突变的引物可以用于血友病病因学分类的应用。
所述血友病病因学分类的步骤如下:
(1)用MC-1000血凝仪检测患者的FⅧ/FⅨ:C,确定是否为FⅧ/FⅨ缺乏。
(2)8对F9引物,以及39对、22倒位3条、1倒位4条F8引物,用ABI 3100基因分析仪测序技术及优选多重与倒转环化PCR扩增技术检测患者的FⅧ/FⅨ基因突变;继用改良Nijmegen法检测患者的FⅧ/FⅨ:Ab。
(3)将检测结果分类为:血友病患者与其母亲的FⅧ/FⅨ基因均发生突变,而且突变的位点及类型完全相同,则为先天遗传性;血友病患者的FⅧ/FⅨ基因突变,而其母亲的FⅧ/FⅨ基因正常,且无家族遗传史,则为先天非遗传性;血友病患者及其母亲的FⅧ/FⅨ基因均正常,且无家族遗传史,血友病患者无既往异常出血史,但FⅧ/FⅨ:Ab检测常为阳性,则为后天获得性。
根据血友病病因学分类,可以应用非因子药物对血友病进行治疗,可使血友病患者由重型转变为中型,轻型,后天获得者FⅧ/FⅨ:C可恢复正常,进一步验证本发明的血友病病因学分类方法确切、可靠,为预防血友病出血和避免残疾,开辟了一条新思路、新途径。