Viral Hepatitis Laboratory, Oswaldo Cruz Institute, Fundação Oswaldo Cruz (Fiocruz, Rio de Janeiro, Brazil.
Departamento de Educação, Instituto Federal de Educação, Ciência e Tecnologia do Ceará, Fortaleza, Brazil.
Microbiologyopen. 2021 Mar;10(2):e1161. doi: 10.1002/mbo3.1161.
The dried blood spot (DBS) samples are a useful resource for viral DNA isolation and important in increasing access to HBV diagnosis. However, the choice of the DNA extraction method is crucial for reliable results. We compared the reliability of four DNA extraction methods using DBS samples for the qualitative and quantitative detection of HBV. A panel of serially diluted HBV DNA in whole blood was spotted onto filter paper (Whatman 903 paper and Whatman FTA cards). Four methods were used to extract DNA: QIAamp DNA Blood Mini Kit (Qiagen); High Pure Viral Nucleic Acid Kit (Roche); Invisorb Spin Blood Midi Kit (Invitek), and DBS Genomic DNA Isolation Kit (Norgen Biotek). Two qualitative PCRs for the core and surface gene regions of HBV were used, and in-house real-time PCR was also evaluated. It was possible to detect HBV DNA using all extraction and PCR protocols. The lowest limit of detection was found using Whatman 903 paper, Roche extraction, and qualitative PCR (20 copies of HBV DNA per ml) for the surface/polymerase HBV region. In the case of in-house real-time PCR, the lowest limit of detection was found using both Roche and Qiagen assays (estimated 2 × 10 copies per ml). These results suggest the importance of both the extraction method and PCR protocol in detecting HBV DNA in DBS. This study provides insights into the utility of DBS samples in HBV molecular diagnosis and their feasibility in low resource areas where cold storage and transportation may be difficult.
干血斑 (DBS) 样本是用于病毒 DNA 分离的有用资源，对于增加乙型肝炎病毒 (HBV) 诊断的可及性非常重要。然而，DNA 提取方法的选择对于获得可靠的结果至关重要。我们比较了四种使用 DBS 样本提取 DNA 的方法的可靠性，用于 HBV 的定性和定量检测。将一系列稀释的 HBV DNA 全血斑点涂在滤纸上（Whatman 903 滤纸和 Whatman FTA 卡）。使用四种方法提取 DNA：Qiagen 的 QIAamp DNA 血液迷你试剂盒；Roche 的 High Pure Viral Nucleic Acid 试剂盒；Invitek 的 Invisorb Spin Blood Midi 试剂盒和 Norgen Biotek 的 DBS 基因组 DNA 分离试剂盒。使用两种用于 HBV 核心和表面基因区域的定性 PCR，以及内部实时 PCR 进行评估。使用所有提取和 PCR 方案都可以检测到 HBV DNA。使用 Whatman 903 滤纸、罗氏提取和定性 PCR（HBV 表面/聚合酶区域每毫升 20 个 HBV DNA 拷贝）可以检测到最低检测限。对于内部实时 PCR，罗氏和 Qiagen 测定均发现最低检测限（估计每毫升 2×10 拷贝）。这些结果表明，提取方法和 PCR 方案在 DBS 中检测 HBV DNA 都很重要。本研究提供了关于 DBS 样本在 HBV 分子诊断中的应用以及在可能难以进行冷藏和运输的资源有限地区的可行性的见解。
