基于聚合酶链反应的乙型肝炎病毒DNA实时检测和定量
Summary
基于实时聚合酶链反应 （PCR） 的乙型肝炎病毒 （HBV） DNA 检测和定量是一种诊断和监测 HBV 感染的灵敏且准确的方法。在这里，我们提出了一种用于HBV DNA检测和样本病毒载量测量的方案。
Abstract
乙型肝炎病毒（HBV）是全球肝病的重要原因。它可能导致急性或慢性感染，使个人极易患致命性肝硬化和肝癌。准确检测和定量血液中的HBV DNA对于诊断和监测HBV感染至关重要。检测HBV DNA的最常用方法是实时荧光定量PCR，可用于检测病毒并评估病毒载量，以监测对抗病毒治疗的反应。在这里，我们描述了使用IVD标记的基于实时PCR的试剂盒检测和定量人血清或血浆中HBV DNA的详细方案。该试剂盒使用靶向 HBV 基因组高度保守的核心区域的引物和探针，可以准确定量所有 HBV 基因型（A、B、C、D、E、F、G、H、I 和 J）。该试剂盒还包括一个内源性内控品，用于监测可能的PCR抑制。该测定运行 40 个循环，其截止值为 38 Ct。 对于临床样品中 HBV DNA 的定量，试剂盒随附一组 5 种定量标准品。这些标准品包含已知浓度的 HBV 特异性 DNA，这些 DNA 是根据 WHO 第 4个 用于核酸检测的 HBV DNA 国际标准（NIBSC 代码 10/266）校准的。这些标准品用于验证HBV特异性DNA扩增的功能，并生成标准曲线，从而可以定量样品中的HBV DNA。使用PCR试剂盒检测低至2.5 IU/mL的HBV DNA。该试剂盒的高灵敏度和可重复性使其成为临床实验室的强大工具，帮助医疗保健专业人员有效诊断和管理 HBV 感染。
Introduction
乙型肝炎病毒 （HBV） 是一种部分双链 DNA 病毒，来自正肝病毒属和肝萘病毒科 1。它可以引发持续一生的慢性感染，可能导致肝硬化和肝细胞癌 2,3,4。根据世界卫生组织 （WHO） 的数据，2019 年估计有 2.96 亿人患有慢性乙型肝炎感染，每年有 150 万人新感染5.
血清或血浆样本中HBV DNA的鉴定和测量是检测持续乙型肝炎感染个体、评估抗病毒治疗疗效和预测治疗成功概率的宝贵方法6,7,8,9,10,11。高病毒载量与肝病进展的风险增加有关，包括肝硬化和肝癌12,13。因此，精确测量病毒载量对于跟踪HBV感染的进展和为治疗决策提供信息至关重要。
与传统的PCR技术相比，实时荧光定量PCR检测具有更高的灵敏度、更宽的动态范围和更准确的HBV DNA定量14,15,16,17。市场上有几种基于实时荧光定量PCR的商业分子诊断试剂盒，用于定量血清或血浆样本中的HBV DNA。在这里，我们描述了使用市售的、带有IVD标记的基于实时荧光定量PCR的试剂盒检测和定量人血清或血浆中HBV DNA的详细工作流程。该试剂盒是一种广泛使用的 18,19、低成本但高灵敏度的检测试剂盒，其性能特征可与其他市售的带有 CE 标志的试剂盒18 相媲美。除了HBV靶区扩增外，试剂盒中还包括一个内源性内控基因，以验证样品质量、提取DNA的质量、PCR扩增和可能的PCR抑制。
Protocol
Representative Results
Discussion
根据供应商信息21，NIBSC 代码 10/266 在 0.5 mL 无核酸酶水中复溶时，单位为 9,55,000 IU/mL。这可以被认为是高负荷HBV阳性样本。本研究中使用的病毒载量试剂盒测定的HBV病毒载量为6,65,900 IU/mL（图4）。由于任何DNA提取试剂盒的DNA提取效率都不是100%，因此在纯化过程中经常会丢失一些DNA。尽管试剂盒确定的病毒载量略低于试剂（NIBSC 代码 10/266）供应商的指定值?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们感谢 Praveen、Kusum、Chandan、Isha、Rashmi、Babli、Shivani、Ankita 和 Shikha 提供的技术援助。
Materials
| 4th WHO International Standard for hepatitis B virus DNA | NIBSC | NIBSC code: 10/266 | The 4th WHO International Standard for hepatitis B virus (HBV) DNA, NIBSC code 10/266, is intended to be used for the calibration of secondary reference reagents used in HBV nucleic acid amplification techniques (NAT). |
| ABI real-time PCR machines | ThermoFisher Scientific | ABI 7500; QuantStudio 5 | This is a real time PCR machine |
| HBV, HCV and HIV Multiplex 14/198 | NIBSC | NIBSC code: 14/198 | This is a very low positive triplex reagent for NAT assays containing hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency virus-1 (HIV-1) |
| QIAGEN real-time PCR machine | Qiagen | Rotor-Gene Q | This is a real time PCR machine |
| TRUPCR HBV Viral Load kit | Kilpest India Limited | 3B294 | This is a real time PCR based kit used in this study for the HBV DNA detection and viral load determination |
| TRUPCR Viral Nucleic Acid Extraction Kit | Kilpest India Limited | 3B214 | This is a DNA extraction kit used for the extraction of HBV viral DNA from NIBSC:10/266 and NIBSC:14/198 |
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