基因芯片检测拉米夫定治疗慢性乙型肝炎后引起的HBV YMDD变异

Detection and identification of emergence of HBV MDD motif mutation during lamivudine treatment by hybridization to an oligonucleotide array

目的 建立乙型肝炎病毒变异基因诊断芯片对拉米夫定治疗慢性乙型肝炎过程中出现的肝炎病毒P基因区YMDD变异进行快速准确的检测诊断方法。方法 设计特异性寡核苷酸探针数组 ,特别处理芯片载体 ,用点样法制备乙型肝炎病毒变异基因诊断芯片。选择 30例服用拉米夫定后 ,HBVDNA转阴 ( <5 0 0copies/ml) ,6 8周后又反跳≥ 5 0 0copies/ml的病人进行基因芯片杂交检测分析。同时用PCR直接测序法对病人血清标本进行双盲HBVDNA聚合酶活性区域测序对照。结果 30例服药后HBVDNA反跳病人中 ,基因芯片测得HBVYMDD变异 2 1例 ,其中YVDD变异11例 ,YIDD变异 10例。HBVDNAPCR直接序列测定结果为有核苷酸A741变为G ,使氨基酸由蛋氨酸 5 5 2变为缬氨酸 (氨基酸基序由YMDD变为YVDD) ,6例核苷酸A669变为C ,氨基酸由亮氨酸5 2 8变为蛋氨酸 ;核苷酸G743 变为T ,使氨基酸由蛋氨酸 5 5 2变为异亮氨酸。 (氨基酸基序由YMDD变为YIDD)。其中有 3例伴有核苷酸T781变为C ,使氨基酸由亮氨酸 5 6 5变为脯氨酸。标本阳性变异序号与基因芯片检测结果完全一致。结论 乙型肝炎病毒变异基因诊断芯片可以同时检测YVDD、YIDD变异 ,与PCR直接测序法比较 ,准确率达 10 0 %。

Objective To set up the quick and exact diagnostic method detecting the HBV P genes YMDD motif mutation occurred during the treatment of chronic hepatitis B with lamivudine by the gene microarrays of HBV dissociation.Methods The DNA microarrays were prepared by spotting fluorescence labeled probes of target genes onto specially lattice glass slides with robotics.For the serum samples of 30 inpatients with hepatitis B of the levels of HBV DNA decreased less than 500 copies/ml after the treatment with lamivudine and jerked more than 500 copies/ml after 68 weeks of the treatment with lamivudine,they were detected for HBV P gene YMDD motif mutation using the double-blind method by gene microarrays and nucleotide sequences assay technique.Results In the 30 patients,there were 21 with HBV P gene YMDD motif mutation including 11 cases of YVDD and 10 cases of YIDD motif mutation detected by the gene microarrays.There were 11 cases of YVDD from YMDD with adenine 741 changed into G cytidine resulted in methionine 552 changed into valine,in which 6 cases with adenine 669 changed into C cytidine and leucine changed into methionine,10 cases of YIDD from YMDD with guanosine 743 altered thymidine resulted in methionine 552 changed into isoleucine,including 3 cases with thymidine 781 changed into C cytidine and leucine 565 altered proline by directly sequencing of PCR products.The positive motif mutation serial number of sample was coincided with the results detected by gene microarrays and nucleotide sequences assay technique.Conclusion The gene microarrays can by used to detect simultaneously HBV YVDD,YIDD motif mutation.The accuracy rate was 100% comparing with nucleotide sequences assay technique.