Huang Zhuhui, Buckwold Victor E
Infectious Disease Research Department, Southern Research Institute, 431 Aviation Way, Frederick, MD 21701, USA.
Mol Cell Probes. 2005 Aug;19(4):282-9. doi: 10.1016/j.mcp.2005.04.004.
Woodchuck hepatitis virus (WHV) is a valuable animal model system for studies of hepatitis B virus infection and accurate assessments of WHV viral load are necessary in these studies. Wild-captured woodchucks that are naturally infected with WHV are sometimes used in these studies, however, the sequence variation in WHV isolates generally precludes the use of TaqMan PCR. To facilitate this, we have created a real-time TaqMan PCR assay for WHV using degenerate primers with inosine residues employed at the locations of known sequence heterogeneity. This TaqMan assay has a dynamic range of 10-10(8) genomic equivalents (ge) of WHV DNA per reaction and the assay is robust and reproducible in the 10(2)-10(7) ge WHV DNA per reaction range (intra-assay coefficient of variation (CV) <2.1%, inter-assay CV <2.9%). During our assay validation, we cloned and analyzed a series of six naturally occurring virus variants that contained sequence heterogeneity in the TaqMan primer sequence region. We showed that the presence of some of these sequence variations prevented the PCR amplification of the target when regular primer sequences were used, while degenerate primer sequences were able to efficiently amplify all tested sequences equally well.
土拨鼠肝炎病毒（WHV）是用于研究乙型肝炎病毒感染的重要动物模型系统，在这些研究中准确评估WHV病毒载量是必要的。有时会使用自然感染WHV的野生捕获土拨鼠进行这些研究，然而，WHV分离株中的序列变异通常使TaqMan PCR无法使用。为便于开展此项研究，我们利用在已知序列异质性位置采用肌苷残基的简并引物，创建了一种针对WHV的实时TaqMan PCR检测方法。该TaqMan检测方法每个反应的动态范围为10 - 10⁸个WHV DNA基因组当量（ge），并且在每个反应10² - 10⁷ ge WHV DNA范围内该检测方法稳定且可重复（批内变异系数（CV）<2.1%，批间CV <2.9%）。在我们的检测验证过程中，我们克隆并分析了一系列六个在TaqMan引物序列区域包含序列异质性的天然存在的病毒变体。我们表明，当使用常规引物序列时，其中一些序列变异的存在会阻止靶标的PCR扩增，而简并引物序列能够同样有效地扩增所有测试序列。
