Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis
Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis
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DOI：
10.1128/jcm.01375-06
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发表时间：
2006-12-01
影响因子：
9.4
通讯作者：
Chang, Ting-Tsung
中科院分区：
文献类型：
--
作者：
Liu, Wen-Chun;Mizokami, Masashi;Chang, Ting-Tsung
病毒滴度和基因型显著决定慢性B型肝炎病毒（HBV）感染的临床结局和抗病毒治疗的反应。开发了一种用于大规模A至G基因分型的方法，同时进行病毒定量。使用杂交探针在LightCycler仪器上进行测定。以两步方式从探针的熔点确定基因型。第1组扩增子区分基因型B、E和F与A、C、D和G，并通过实时PCR同时定量病毒血症。然后使用组2-1扩增子或组2-2扩增子反应混合物的解链曲线分析将这些基因型组区分为单一基因型。HBV DNA定量与Amplicor检测试剂盒一致，在10（2）至10（13）拷贝/ml范围内呈线性。与限制性片段长度多态性方法比较，该方法对441份样本的基因分型准确率为923%。该方法可用于所有基因型存在的地区的HBV DNA的基因分型和定量。
Both the viral titer and the genotype significantly determine clinical outcomes and responses to antiviral treatment in chronic hepatitis B virus (HBV) infection. A method was developed for large-scale A-to-G genotyping with simultaneous viral quantification. The assay was run on a LightCycler instrument using hybridization probes. The genotype was determined from the melting points of the probes in a two-step manner. Set 1 amplicons differentiated genotypes B, E, and F from A, C, D, and G and simultaneously quantified viremia by real-time PCR. Melting curve analysis using the set 2-1 amplicon or the set 2-2 amplicon reaction mixture was then used to differentiate these genotype groups into single genotypes. HBV DNA quantification was consistent with that of the Amplicor assay and linear in a range from 10(2) to 10(13) copies/ml. By comparison with the restriction fragment length polymorphism method, 923% of 441 samples were accurately genotyped by the current assay. The method should be useful for genotyping and quantification of HBV DNA in areas where all genotypes exist.