Single-step real-time PCR to quantify hepatitis B virus and distinguish genotype D from non-D genotypes
Single-step real-time PCR to quantify hepatitis B virus and distinguish genotype D from non-D genotypes
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DOI：
10.1111/j.1365-2893.2010.01308.x
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发表时间：
2011-04-01
影响因子：
2.5
通讯作者：
Tacke, F.
中科院分区：
文献类型：
--
作者：
Amini-Bavil-Olyaee, S.;Pourkarim, M. R.;Tacke, F.
B型肝炎病毒（HBV）病毒载量及其基因型在临床结局、疾病管理和抗病毒治疗反应中起重要作用。在世界许多地区，如欧洲或中东，区分HBV基因型D与非D与治疗决策最相关，因为基因型D感染的患者对基于干扰素的治疗方案反应较差。在这里，我们开发了一种内部实时PCR，以基于熔解曲线分析一致地评估HBV基因型（D与非D）并量化病毒载量。用所有HBV基因型的对照质粒建立基因型区分，并用来自感染六种不同HBV基因型的患者的57份临床样本进行验证。我们的内部实时PCR检测可以区分HBV基因型D从非D使用一步熔解曲线分析与2摄氏度的差异，在所有样品的熔解曲线温度测试。用WHO HBV国际标准品校准病毒载量定量，证明在3.2 x 102至3.2 x 1010 IU/mL的线性范围内与商业试剂盒具有良好的相关性（r = 0.852; P < 0.0001）。总之，我们开发了一种快速，简单和具有成本效益的方法，同时定量和区分HBV基因型D与非D的一步PCR运行和熔解曲线分析。这种检测方法应该是一种有用的诊断替代方法，以帮助临床决定是否开始和选择抗病毒治疗，特别是在HBV基因型D流行率高的地区。
Hepatitis B virus (HBV) viral load and its genotype play important roles in clinical outcome, management of disease and response to antiviral therapy. In many parts of the world such as Europe or the Middle East, distinguishing HBV genotype D from non-D is most relevant for treatment decisions, because genotype D-infected patients respond poorly to interferon-based therapeutic regimens. Here, we developed an in-house real-time PCR to concordantly assess HBV genotype (D vs non-D) based on melt curve analysis and quantify the viral load. Genotype distinction was established with control plasmids of all HBV genotypes and validated with 57 clinical samples from patients infected with six different HBV genotypes. Our in-house real-time PCR assay could discriminate HBV genotype D from non-D using single-step melt curve analysis with a 2 degrees C difference in the melt curve temperature in all samples tested. Viral load quantification was calibrated with the WHO HBV international standard, demonstrating an excellent correlation with a commercial kit (r = 0.852; P < 0.0001) in a linear range from 3.2 x 102 to 3.2 x 1010 IU/mL. In conclusion, we developed a rapid, simple and cost-effective method to simultaneously quantify and distinguish HBV genotypes D from non-D with a single-step PCR run and melt curve analysis. This assay should be a useful diagnostic alternative to aid clinical decisions about initiation and choice of antiviral therapy, especially in geographical regions with a high prevalence of HBV genotype D.