CBF Systems Inc., College Plaza, Edmonton, AB, Canada.
Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, AB, Canada.
PLoS One. 2018 May 10;13(5):e0197100. doi: 10.1371/journal.pone.0197100. eCollection 2018.
In this manuscript, we report the design and development of a fast, reliable instrument to run gel-based cassette polymerase chain reactions (PCR). Here termed the GelCycler Mark II, our instrument is a miniaturized molecular testing system that is fast, low cost and sensitive. Cassette PCR utilizes capillary reaction units that carry all reagents needed for PCR, including primers and Taq polymerase, except the sample, which is loaded at the time of testing. Cassette PCR carries out real time quantitative PCR followed by melt curve analysis (MCA) to verify amplicon identity at the expected melt temperature (Tm). The cassette PCR technology is well developed, particularly for detecting pathogens, and has been rigorously validated for detecting pathogenic Escherichia coli in meat samples. However, the work has been hindered by the lack of a robust and stable instrument to carry out the PCR, which requires fast and accurate temperature regulation, improved light delivery and fluorescent recording, and faster PCR reactions that maintain a high sensitivity of detection. Here, we report design and testing of a new instrument to address these shortcomings and to enable standardized testing by cassette PCR and commercial manufacture of a robust and accurate instrument that can be mass produced to deliver consistent performance. As a corollary to our new instrument development, we also report the use of an improved design approach using a machined aluminum cassette to meet the new instrument standards, prevent any light bleed across different trenches in each cassette, and allow testing of a larger number of samples for more targets in a single run. The GelCycler Mark II can detect and report E. coli contamination in 41 minutes. Sample positives are defined in as having a melt curve comparable to the internal positive control, with peak height exceeding that of the internal negative control. In a fractional analysis, as little as 1 bacterium per capillary reaction unit is directly detectable, with no enrichment step, in 35 cycles of PCR/MCA, in a total time of 53 minutes, making this instrument and technology among the very best for speed and sensitivity in screening food for pathogenic contamination.
在本手稿中，我们报告了一种快速、可靠的凝胶盒式聚合酶链反应（PCR）仪器的设计和开发。我们称这种仪器为 GelCycler Mark II，它是一种小型化的分子测试系统，快速、低成本且灵敏。盒式 PCR 利用毛细管反应单元，这些单元携带 PCR 所需的所有试剂，包括引物和 Taq 聚合酶，但样本除外，样本在测试时加载。盒式 PCR 进行实时定量 PCR 后进行熔解曲线分析（MCA），以在预期熔解温度（Tm）下验证扩增子的身份。盒式 PCR 技术已经很成熟，特别是用于检测病原体，并且已经经过严格验证，可以检测肉样中的致病性大肠杆菌。然而，这项工作受到缺乏强大稳定的仪器来进行 PCR 的阻碍，PCR 需要快速准确的温度调节、改进的光传输和荧光记录，以及保持高检测灵敏度的更快 PCR 反应。在这里，我们报告了一种新仪器的设计和测试，以解决这些缺点，并使盒式 PCR 标准化测试和制造稳健、准确的仪器成为可能，这种仪器可以大规模生产，以提供一致的性能。作为我们新仪器开发的推论，我们还报告了使用改进的设计方法，使用机械加工的铝制盒式来满足新仪器标准，防止每个盒式中的不同沟槽之间的任何光线泄漏，并允许在单个运行中测试更多目标的更多样本。GelCycler Mark II 可以在 41 分钟内检测和报告大肠杆菌污染。样本阳性的定义为熔解曲线与内部阳性对照相似，峰高超过内部阴性对照。在分数分析中，在没有富集步骤的情况下，每个毛细管反应单元直接检测到低至 1 个细菌，在 35 个 PCR/MCA 循环中，总时间为 53 分钟，使该仪器和技术成为速度和灵敏度最高的用于筛选食物中致病污染的仪器和技术之一。
