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目的比较DNA序列测定法与实时荧光法检测YMDD突变的结果,并对除YMDD突变之外的耐药相关基因突变及其意义进行讨论。方法收集89例慢性乙肝患者的92份血清样本,均用实时荧光PCR法做YMDD突变检测。采用巢式PCR法扩增HBV反转录酶(RT)基因,对PCR产物进行DNA双向测序,采用NTI软件比对结果,并对RT基因上其他11个已知耐药相关突变位点进行分析。结果在37份YMDD突变阴性的样本中,DNA测序法检测结果为33份M204M(未突变)、1份M204I、3份M204V;4份YMDD突变阳性样本均为突变/未突变序列共存;另检出7份样本存在ADV耐药相关突变,占18.9%(7/37)。在55份YMDD突变阳性样本中,DNA测序法检测到52份,阳性结果符合率为94.5%(52/55);另检出5例存在ADV或ETV耐药相关突变,占9.1%(5/55)。结论DNA序列测定法检测HBVRT基因耐药相关突变敏感性高,重复性好,与实时荧光PCR方法的检测结果有较高的符合率,可同时检出YMDD突变以外的各种耐药相关突变,有助于临床全面了解患者对核苷(酸)类似物的耐药情况,合理地制订抗HBV治疗方案。
Objective To compare the results of YMDD mutation detection by DNA sequencing and real-time fluorescence assay, and to explore the significance of YMDD resistance mutations and their significance. Methods Ninety-two serum samples of 89 patients with chronic hepatitis B were collected and detected by YMDD mutation using real-time fluorescence PCR. The HBV reverse transcriptase (RT) gene was amplified by nested PCR and the DNA was sequenced by PCR. The results of NTI software were compared and the other 11 known mutations in RT gene were analyzed . Results In 37 samples with negative YMDD mutation, 33 M204M (unmutated), 1 M204I and 3 M204V were detected by DNA sequencing. The positive samples of 4 YMDD mutations were both co-existed with mutation / non-mutation. Seven out of seven samples were associated with ADV resistance, accounting for 18.9% (7/37). Among 55 samples positive for YMDD mutation, 52 were detected by DNA sequencing, and the positive rate was 94.5% (52/55). In addition, 5 cases were detected with ADV or ETV resistant mutations, accounting for 9.1% (5 / 55). Conclusion DNA sequencing has high sensitivity and repeatability in detecting drug-resistant mutations of HBVRT gene, and has high coincidence rate with real-time PCR. It can detect all kinds of resistance-related mutations besides YMDD mutation, Help clinical fully understand patients with nucleotide (acid) analogs resistance, rational development of anti-HBV treatment options.