基因芯片检测HBV DNA聚合酶区基因多态性

Detection of polymorphism of hepatitis B virus P gene by DNA microarray

目的 :建立基因芯片检测HBVDNA聚合酶区基因多态性。方法 :PCR扩增HBVDNAP区nt45 5～ 796片断 ,与预制的基因芯片杂交 ,运用激光共聚焦荧光检测系统扫描芯片分析基因突变类型。结果 :芯片检测HBVDNAP区变异具有较好的特异性和准确性 ;初步应用显示 :慢性乙肝组nt5 2 8和nt5 5 2位点突变率分别为 8.6%和 9.6% ,而无症状肝炎组和重症肝炎组突变率均为 0 ;拉米夫定治疗组与非拉米夫定治疗组比较 ,nt5 2 8突变率分别为 10 .5 %和 8.1% ,nt5 5 2突变率分别为 15 .8%和 8.1%。结论 :HBVDNA聚合酶区基因多态性分布与病情轻重无关 ,而与病程慢性迁延有关 ;拉米夫定在耐药突变中发挥了重要作用 ;基因芯片检测乙肝患者耐药基因有其临床应用价值。

Objective:To detect HBV P gene polymorphism by DNA microarray . Methods:The nt455～796 segment of HBV DNA P gene were hybridized with oligonucleotide probes ,which were pre-stablized on the chip by Cartesian Pixsys 7500. The microchip were scanned with General Scanning 3000 . According to the corresponding probe sequence, mutation type can be determined .Results:The preliminary experiment showed that the assay was sensitive and specific. The mutation frequency of HBV P gene are 8.6% (nt528) and 9.6% (nt552 ) in chronic hepatitis B , and 0.00% (nt528/552) in asymptomatic carrier and severe hepatitis B , respectively . Nt528 mutation frequency of HBV P gene are 10.5% in the patients with lamivudine treatment and 8.1% in the patients without lamivudine treatment. Nt522 mutation frequency of HBV P gene are 15.8% in the patients with lamivudine treatment and 8.1% in the patients without lamivudine treatment .Conclusions:The mutation frequency of HBV P gene(nt528/552 )is relevant to chronic programming of hepatitis B .The lamivudine treatment play a important role in the sites mutation . The microarray of lamivudine resistence related mutation may be useful for clinical treatment of the patients with hepatitis B.