目的 建立一种基于非标记探针高分辨率熔解曲线技术(HRM)检测HBV P区rt204位点耐药突变的方法.方法 构建野生株rt204M、突变株rt204I和rt204V的质粒,设计、优化探针.利用质粒作为标准品建立HRM特征性解链图谱.收集2010年5月至2012年5月宁波大学附属医院诊治的185例慢性乙型肝炎患者外周血样本共185份,用HRM技术检测所有样本,并与特征性解链图谱比对,筛选出rt204位点的突变,再经基因测序验证.采用配对x2检验比较两种方法的变异检出率.结果 突变株rt204I、rt204V和野生株rt204M的熔解温度(Tm)分别为58.0℃、60.6℃和62.5℃；185份样本中,HRM技术成功分析168份(90.8％),基因测序成功分析155份(83.8％),二者差异有统计学意义(P＜0.01)；同时被两种方法检出的155份临床样本中,非标记探针HRM技术检测到野生株75份(rt204M),变异株80份(55份rt204I、25份rt204V),变异检出率为51.6％(80/155)；基因测序法检测到野生株110份(rt204M),变异株45份(30份rt204I、15份rt204V),变异检出率为29.0％ (45/155),两种方法差异有统计学意义(P＜0.01).结论 非标记探针HRM技术是一种简单、灵敏、快速、特异的HBV P区rt204位点耐药突变检测方法.%Objective To establish a method of high resolution melting analysis (HRM) with unlabeled probe for detection of rt204 mutation in HBV P gene.Methods Plasmids with wild strain rt204M,mutant strains rt204I and rt204V were constructed,and the probes were designed and optimized.HRM plots were established by the constructed plasmids.A total of 185 samples were collected from patients with chronic hepatitis B in the Affiliated Hospital of School of Medicine of Ningbo University during May 2010 and May 2012.All samples were detected by HRM,and matched with characteristic melting pattern.rt204 mutations were screened,and then verified by DNA sequencing.Paired x2 test was used for the comparison of the detection of mutations.Results The melting temperatures for rt204I,rt204V and rt204M were 58.0℃,60.6℃ and 62.5℃,respectively.Among 185 samples,168 samples (90.8％) could be analyzed by HRM method,and 155 samples (83.8％) coule be successfully sequenced (P ＜0.01).In the 155 samples which were completely analyzed by HRM assay and sequencing,75 samples were rt204M,55samples were rt204I,and 25 samples were rt204V by using HRM method,with an overall mutation detection rate of 51.6％ (80/155) ; and by sequencing,110 samples were rt204M,30 samples were rt204I,and 15samples were rt204V,with an overall mutation detection rate of 29.0％ (45/155).The difference onmutation rates detected by the above two methods was of statistical significance (P ＜ 0.01).Conclusion HRM with unlabeled probe is simple,sensitive,rapid and specific for detection of rt204 mutation in HBV P gene.
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