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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 establish a diagnostic chip for hepatitis B virus (HBV) mutation gene and to detect the YMDD mutation of P gene in hepatitis B virus during lamivudine treatment for rapid and accurate diagnosis. Methods: Design specific oligonucleotide probes. Preparation of HBV Mutation Gene Diagnostic Chips by Dot Specimen. Thirty patients admitted to hospital with lamivudine were tested for gene chip hybridization in HBVDNA-negative patients (> 500 copies / ops) for a further 68 weeks and again> 5000copies / ml. Meanwhile, PCR-direct sequencing was performed on 30 patients with serum samples for double-blind HBVDNA polymerase region sequencing. Results: Twenty-one HBVYMDD mutations were detected in gene chips of 30 patients with HBVDNA rebound after medication, of which 11 were YVDD and 10 were YIDD. Direct sequencing revealed that nucleotide A741 changed to G, amino acid changed from methionine 552 to valine (amino acid motif changed from YMDD to YVDD), and 6 nucleotides A669 changed to C, Leucine 528 to methionine, nucleotide G743 to T, and amino acid methionine 552 to isoleucine (amino acid motif changed from YMDD to YIDD). Three of them were accompanied by nucleotide T781 becoming C, which changed the amino acid from leucine 565 to proline. The result is exactly the same as the gene chip. Conclusion: The HBV variant gene diagnostic chip can detect both YVDD and YIDD mutations at the same time. The accuracy rate of PCR is 100% with no false positive.