Laboratory for Sensors, Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Köhler-Allee 103, Freiburg, Germany.
Analyst. 2020 Apr 7;145(7):2554-2561. doi: 10.1039/c9an02333j. Epub 2020 Feb 19.
Nucleic acid amplification techniques such as real-time PCR are essential instruments for the identification and quantification of viruses. They are fast, very sensitive and highly specific, but often require elaborate and labor intensive sample preparation to achieve successful amplification of the target sequence. In this work we demonstrate the complete microfluidic preparation of amplifiable virus DNA from dilute specimens. Our approach combines free-flow electrophoretic preconcentration of viral particles with thermal lysis and gel-electrophoretic nucleic acid extraction on a single device. The on-chip preconcentration achieves a capture efficiency of >99% for dilute suspensions of bacteriophage PhiX174. Following preconcentration, phages are thermally lysed and released DNA is recovered after 40 s of on-chip gel-electrophoresis with a recovery rate of ∼73%. Furthermore we demonstrate a detection limit of ∼1 PFU ml (∼0.02 DNA copies per μl) for the detection of bacteriophage PhiX174 by PCR. To simplify operation of the device, we describe the development of a custom-made chip holder as well as a compact peristaltic pump and power supply, which enable user-friendly operation with low risk of cross-contamination and high potential for automation in the field of point-of-care diagnostics.
核酸扩增技术，如实时 PCR，是鉴定和定量病毒的重要工具。它们快速、非常敏感且高度特异性，但通常需要精心设计和劳动密集型的样本制备，以实现目标序列的成功扩增。在这项工作中，我们展示了从稀释样本中完全制备可扩增病毒 DNA 的微流控方法。我们的方法将病毒颗粒的自由流电泳预浓缩与热裂解和凝胶电泳核酸提取结合在一个装置上。在芯片上预浓缩，对稀菌噬菌体 PhiX174 悬浮液的捕获效率>99%。预浓缩后，噬菌体被热裂解，释放的 DNA 在芯片上凝胶电泳 40 秒后回收，回收率约为 73%。此外，我们通过 PCR 检测到噬菌体 PhiX174 的检测限约为 1 PFU ml（约 0.02 个 DNA 拷贝/μl）。为了简化设备的操作，我们描述了一种定制芯片支架以及一种紧凑的蠕动泵和电源的开发，这使得设备具有用户友好的操作、低交叉污染风险和在即时诊断领域实现自动化的高潜力。
