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  

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

作　　者：曹新民[1] 赵伟[1] 刘伟[1] 刘全俊[2] 张林[1] 张汉荣[1] 刘新珏[1] 周镇先[1] 

机构地区：[1]东南大学医学院附属南京市第二医院肝内科,江苏南京210003 [2]东南大学分子与生物电子国家重点实验室,江苏南京210096

出　　处：《南京医科大学学报（自然科学版）》2005年第9期637-640,共4页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省科技厅重点资助项目;江苏省卫生厅重点资助项目(BS2000028)

摘　　要：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。Objective： To set up using the gene chip technology to detect and identify quickly and accurately 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 of 30 patients with hepatitis B after 68 weeks treated 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： By the gene chips there were 21 patients with HBV P gene YMDD motif mutation including II cases with YVDD and I0 cases with YIDD motif mutation.By directly sequencing of PCR products there were 11 cases of YVDD with adenine ^741 changed into cytidine resulted in methionine ^552 changed into valine in which 6 cases with adenine ^669 changed into cytidine and leucine changed into methionine I0 cases of YIDD motif mutation with guanosine ^743 altered thymidine methionine ^552 changed into isoleucine including 3 cases with thymidine ^281 changed into cytidine and leucine ^565 altered proline. Conclusion： The gene chip can be used to test HBV YVDD,YIDD motif mutation compaed with nucleotide sequences assay technique has a 100% accuracy rate.

关 键 词：基因芯片 拉米夫定 病毒变异 乙型肝炎病毒 

分 类 号：R575.2[医药卫生—消化系统]