基因芯片检测拉米夫定引起的乙型肝炎病毒基因YMDD变异的研究

目的:建立乙型肝炎病毒(HBV)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。用点样法制备HBV变异基因诊断芯片。选择住院患者30例服用拉米夫定后,HBVDNA转阴[<500拷贝(opies/ml)]68周后又反跳≥5000copies/ml的患者进行基因芯片杂交检测。同时用PCR直接测序法对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HBVYMDD变异21例,其中YVDD变异11例,YIDD变异10例。直接序列测定发现有核苷酸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,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。其结果与基因芯片完全一致。结论:HBV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。

To study the detection of emergence of HBV YMDD motif mutation caused by lamivudine by the gene chip

Objective: To set up using the gene chip technology to detect and identify quickly and accurately the HBV P geneYMDD motif mutation during the chronic hepatitis treated with lamivudine. Methods: DNA microarrays were prepared by spottingfluorescence labeled probes of target genes onto specially lattice glass slides with robotics. The serum samples of 30 patients withhepatitis B after 68 weeks treated with lamivudine were detected double-blind by gene chips and by nucleotide sequences assaytechnique to identify the rate of emergence of HBV P gene YMDD motif mutation. Results: By the gene chips there were 21 patientswith HBV P gene YMDD motif mutation including 11 cases with YVDD and 10 cases with YIDD motif mutation.By directlysequencing of PCR products there were 11 cases of YVDD with adenine 741 changed into cytidine resulted in methionine 552 changedinto valine in which 6 cases with adenine 669 changed into cytidine and leucine changed into methionine 10 cases of YIDD motifmutation with guanosine743 altered thymidine methionine 552 changed into isoleucine including 3 cases with thymidine 281 changed intocytidine and leucine 565 altered proline. Conclusion: The gene chip can be used to test HBV YVDD, YIDD motif mutation compaedwith nucleotide sequences assay technique has a 100% accuracy rate.