The transmission of bloodborne viruses through transfusion remains a major blood supply–related safety concern, with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) being the most important pathogens in this context. Real-time bioluminescent pyrophosphate testing has been developed as a means of readily detecting bacterial cells within particular sample types without requiring the use of expensive or complex instrumentation. The sensitivity of this approach, however, is often limited such that it is not compatible with many potential applications. In this study, we sought to overcome the limitations of this pyrophosphate bioluminescent assay format by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) in place of dATP for PCR amplification, thereby dramatically reducing background signal levels. We leveraged this combination PCR and bioluminescent pyrophosphate assay approach to facilitate HBV detection. This assay yielded a limit of detection of 500 copies/mL, making it more sensitive than traditional bioluminescent assays, about 1000 times more sensitive than that of PCR product analysis by agarose gel electrophoresis, and roughly as sensitive as qPCR as a means of detecting viral DNA. We then used this assay to analyze 100 serum samples, with qPCR being used for result validation. The assay required 100 min to complete, and was able to detect as few as 500 copies/mL of viral DNA. Overall, our approach was rapid, sensitive, and simple, enabling users to readily detect HBV in a reliable and efficient manner.

通过输血传播血源性病毒仍然是与血液供应相关的主要安全问题，其中乙型肝炎病毒（HBV）、丙型肝炎病毒（HCV）和人类免疫缺陷病毒（HIV）是这方​​面最重要的病原体。实时生物发光焦磷酸盐测试已被开发为一种无需使用昂贵或复杂仪器即可轻松检测特定样品类型中细菌细胞的方法。然而，这种方法的灵敏度通常是有限的，因此它与许多潜在的应用不兼容。在本研究中，我们试图通过使用 2-脱氧腺苷-5-(α-硫代)-三磷酸 (dATPαS) 代替 dATP 进行 PCR 扩增来克服这种焦磷酸生物发光测定形式的局限性，从而显着降低背景信号水平。我们利用这种组合 PCR 和生物发光焦磷酸测定方法来促进 HBV 检测。该检测的检测限为 500 拷贝/mL，比传统生物发光检测更灵敏，比琼脂糖凝胶电泳 PCR 产物分析灵敏约 1000 倍，与病毒检测方法 qPCR 大致一样灵敏脱氧核糖核酸。然后，我们使用该测定法分析 100 个血清样本，并使用 qPCR 进行结果验证。该检测需要 100 分钟才能完成，并且能够检测到低至 500 个拷贝/mL 的病毒 DNA。总体而言，我们的方法快速、灵敏且简单，使用户能够以可靠且高效的方式轻松检测 HBV。