University of Utah, Rm 5R441, 1795 E South Campus Dr., Salt Lake City, Utah 84112, USA.
Anal Chem. 2010 Feb 15;82(4):1546-50. doi: 10.1021/ac902398c.
An inexpensive plastic disk disposable was designed for digital polymerase chain reaction (PCR) applications with a microfluidic architecture that passively compartmentalizes a sample into 1000 nanoliter-sized wells by centrifugation. Well volumes of 33 nL were attained with a 16% volume coefficient of variation (CV). A rapid air thermocycler with aggregate real-time fluorescence detection was used, achieving PCR cycle times of 33 s and 94% PCR efficiency, with a melting curve to validate product specificity. A CCD camera acquired a fluorescent image of the disk following PCR, and the well intensity frequency distribution and Poisson distribution statistics were used to count the positive wells on the disk to determine the number of template molecules amplified. A 300 bp plasmid DNA product was amplified within the disk and analyzed in 50 min with 58-1000 wells containing plasmid template. Target concentrations measured by the spinning disk platform were 3 times less than that predicted by absorbance measurements. The spinning disk platform reduces disposable cost, instrument complexity, and thermocycling time compared to other current digital PCR platforms.
一种廉价的塑料圆盘被设计用于数字聚合酶链反应（PCR）应用，其微流控结构通过离心将样品被动分隔成 1000 个纳升大小的孔。孔体积为 33nL，体积变化系数为 16%。使用具有综合实时荧光检测功能的快速空气热循环仪，实现了 33 秒的 PCR 循环时间和 94%的 PCR 效率，并采用熔解曲线验证了产物特异性。PCR 后，CCD 相机获取圆盘的荧光图像，并使用孔强度频率分布和泊松分布统计数据来计算圆盘上的阳性孔数，以确定扩增的模板分子数。在圆盘内扩增了 300bp 的质粒 DNA 产物，在 50 分钟内分析了 58-1000 个含有质粒模板的孔。通过旋转圆盘平台测量的目标浓度比吸光度测量预测的低 3 倍。与其他当前的数字 PCR 平台相比，旋转圆盘平台降低了一次性成本、仪器复杂性和热循环时间。
