Hurth Cedric, Yang Jianing, Barrett Matthew, Brooks Carla, Nordquist Alan, Smith Stanley, Zenhausern Frederic
Center for Applied Nanobioscience & Medicine, The University of Arizona College of Medicine, 425 N 5th Street, Phoenix, AZ, 85004, USA,
Biomed Microdevices. 2014 Dec;16(6):905-14. doi: 10.1007/s10544-014-9895-8.
We report a microfluidic device and measurement method to perform real-time PCR (or qPCR) in a miniaturized configuration for on-chip implementation using reaction volumes of less than 20 μL. The qPCR bioreactor is designed as a module to be embedded in an automated sample-in/profile-out system for rapid DNA biometrics or human identification. The PCR mixture is excited with a 505 nm diode-pumped solid-state laser (DPSSL) and the fluorescence build-up is measured using optical fibers directly embedded to the sidewalls of the microfluidic qPCR bioreactor. We discuss manufacturing and operating parameters necessary to adjust the internal surface conditions and temperature profiles of the bioreactor and to optimize the yield and quality of the PCR reaction for the amplification of 62 bp hTERT intron fragments using the commercial Quantifiler® kit (Life Technologies, Carlsbad, CA) commonly accepted for genotyping analysis. We designed a microfluidic device suitable for continuously processing a specimen by efficiently mixing the reagents from the kit to a set volume of DNA template on chip. Our approach relies on a calibration curve for the specific device using control DNA. We successfully applied this method to determine the concentration of genomic DNA extracted from a buccal swab on separate microfluidic devices which are operated upstream the qPCR device and perform buccal swab lysis and buccal DNA extraction. A precise correlation between the amount determined on chip and that obtained using a commercial cycler is demonstrated.
我们报告了一种微流控装置和测量方法，可在小型化配置中进行实时聚合酶链反应（或定量聚合酶链反应），以在芯片上实现，反应体积小于20微升。定量聚合酶链反应生物反应器被设计为一个模块，可嵌入自动进样/出样系统，用于快速DNA生物特征识别或身份鉴定。用505纳米二极管泵浦固态激光器（DPSSL）激发聚合酶链反应混合物，并使用直接嵌入微流控定量聚合酶链反应生物反应器侧壁的光纤测量荧光积累。我们讨论了调整生物反应器内表面条件和温度分布以及优化聚合酶链反应产量和质量所需的制造和操作参数，以使用常用于基因分型分析的商业Quantifiler®试剂盒（生命技术公司，加利福尼亚州卡尔斯巴德）扩增62碱基对人端粒酶逆转录酶内含子片段。我们设计了一种微流控装置，通过在芯片上有效地将试剂盒中的试剂与一定体积的DNA模板混合，适用于连续处理样本。我们的方法依赖于使用对照DNA的特定装置的校准曲线。我们成功地应用此方法在单独的微流控装置上测定从口腔拭子中提取的基因组DNA的浓度，这些装置在定量聚合酶链反应装置上游运行，进行口腔拭子裂解和口腔DNA提取。证明了芯片上测定的量与使用商业循环仪获得的量之间的精确相关性。
