Selck David A, Ismagilov Rustem F
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA, United States of America.
PLoS One. 2016 Oct 19;11(10):e0163060. doi: 10.1371/journal.pone.0163060. eCollection 2016.
Nucleic acid amplification tests that are coupled with a digital readout enable the absolute quantification of single molecules, even at ultralow concentrations. Digital methods are robust, versatile and compatible with many amplification chemistries including isothermal amplification, making them particularly invaluable to assays that require sensitive detection, such as the quantification of viral load in occult infections or detection of sparse amounts of DNA from forensic samples. A number of microfluidic platforms are being developed for carrying out digital amplification. However, the mechanistic investigation and optimization of digital assays has been limited by the lack of real-time kinetic information about which factors affect the digital efficiency and analytical sensitivity of a reaction. Commercially available instruments that are capable of tracking digital reactions in real-time are restricted to only a small number of device types and sample-preparation strategies. Thus, most researchers who wish to develop, study, or optimize digital assays rely on the rate of the amplification reaction when performed in a bulk experiment, which is now recognized as an unreliable predictor of digital efficiency. To expand our ability to study how digital reactions proceed in real-time and enable us to optimize both the digital efficiency and analytical sensitivity of digital assays, we built a custom large-format digital real-time amplification instrument that can accommodate a wide variety of devices, amplification chemistries and sample-handling conditions. Herein, we validate this instrument, we provide detailed schematics that will enable others to build their own custom instruments, and we include a complete custom software suite to collect and analyze the data retrieved from the instrument. We believe assay optimizations enabled by this instrument will improve the current limits of nucleic acid detection and quantification, improving our fundamental understanding of single-molecule reactions and providing advancements in practical applications such as medical diagnostics, forensics and environmental sampling.
结合数字读数的核酸扩增测试能够对单分子进行绝对定量，即使在超低浓度下也能实现。数字方法稳健、通用，并且与包括等温扩增在内的多种扩增化学方法兼容，这使得它们对于需要灵敏检测的分析尤其宝贵，例如隐匿感染中病毒载量的定量分析或法医样本中少量DNA的检测。目前正在开发多种微流控平台来进行数字扩增。然而，由于缺乏关于哪些因素会影响反应的数字效率和分析灵敏度的实时动力学信息，数字分析的机理研究和优化受到了限制。能够实时跟踪数字反应的市售仪器仅限于少数几种设备类型和样品制备策略。因此，大多数希望开发、研究或优化数字分析的研究人员依赖于在批量实验中进行的扩增反应速率，而现在人们认识到这是数字效率的不可靠预测指标。为了扩展我们实时研究数字反应如何进行的能力，并使我们能够优化数字分析的数字效率和分析灵敏度，我们构建了一台定制的大幅面数字实时扩增仪器，它可以适应多种设备、扩增化学方法和样品处理条件。在此，我们对该仪器进行了验证，提供了详细的原理图以便其他人能够构建自己的定制仪器，并且我们还包括一套完整的定制软件套件来收集和分析从仪器中检索到的数据。我们相信，该仪器实现的分析优化将提高核酸检测和定量的当前极限，增进我们对单分子反应的基本理解，并在医学诊断、法医和环境采样等实际应用中取得进展。
