基因芯片检测拉米夫定治疗慢性乙型肝炎过程中HBV变异技术的建立和应用

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。方法设计特异性寡核苷酸探针阵列,特殊处理芯片载体。用点样法制备乙型肝炎病毒变异基因诊断芯片。在本院住院治疗患者中选择30例服用拉米夫定后,HBV DNA转阴(<500 copies/ml)168周后又反跳≥5 000 copies/ml的患者进行基因芯片杂交检测分析。同时用PCR直接测序法对上述30例患者血清标本进行双盲HBVDNA聚合酶活性区域测序对照。结果 30例服药后HBVDNA反跳患者中基因芯片测得HBV YMDD变异21例,其中YVDD变异11例。YIDD变异10例。HBVDNA PCR直接序列测定结果,有核苷酸A741变为G,使氨基酸由蛋氨酸552变为缬氨酸(氨基酸基序由YMDD变为YVDD)11例,并有6例核苷酸A669变为C,使氨基酸由亮氨酸528变为蛋氨酸。核苷酸G743变为T,使氨基酸由蛋氨酸552变为异亮氨酸(氨基酸基序由YMDD变为YIDD)10例。其中有3例伴有核苷酸T781变为C,使氨基酸由亮氨酸565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,同PCR直接测序法比较,准确率达100％,无假阳性。

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

Objective Gene chip technology was set up to quickly and accurately detect and identify the HBV P gene YMDD motif mutation during the chronic hepatitis treated with lamivudine. Methods DNA microarrays were prepared by spotting fluorescence labeled probes of target genes onto specially lattice glass slides with robotics. The serum samples from 30 patients with hepatitis B after 68 -week treatment with lamivudine were detected double-blind by gene chips and by nucleotide sequences assay technique to identify the rate of emergence of HBV P gene YMDD motif mutation. Results Twenty-one patients were found HBV P gene YMDD motif mutation by the gene chips including 11 cases with YVDD and 10 cases with YIDD motif mutation. By direct sequencing of the PCR products, 11 cases were found to have YVDD with adenine741 changed into cytidine resulted in methionine552 changed into valine in which 6 cases with adenine669 changed into cytidine and leucine changed into methionine, 10 cases had YIDD motif mutation with guanosine743 altered thymidine methionine changed into isoleucine, including 3 cases with thymidine changed into cytidine and leucine565 altered proline. Conclusion The gene chip can be used to test HBV YVDD, YIDD motif mutation compared with nucleotide sequences assay technique, the accuracy rate was 100% .