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首页> 外文期刊>RSC Advances >Centrifugo-thermopneumatic fluid control for valving and aliquoting applied to multiplex real-time PCR on off-the-shelf centrifugal thermocycler
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Centrifugo-thermopneumatic fluid control for valving and aliquoting applied to multiplex real-time PCR on off-the-shelf centrifugal thermocycler

机译：Centrifugo-Thermopnatic液体控制用于阀门的阀门和等分试样，以在现成的离心式热循环热循环中的多重实时PCR

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We introduce microfluidic automation of geometrically multiplexed real-time PCR to off-the-shelf Rotor-Gene Q thermocyclers (RGQ, QIAGEN GmbH, Hilden, Germany). For centrifugal fluid control the RGQ provides low and constant rotation of 400 rpm, only. Compatibility to this very limited flexibility of centrifugal actuation is achieved by using thermal gas compression and expansion for valving and aliquoting. In contrast to existing thermo-pneumatic actuation, centrifugo-thermopneumatic (CTP) fluid control employs the induced change of partial vapor pressure by global temperature control as actuation parameter for two new unit operations: CTP siphon valving and CTP two-stage aliquoting. CTP siphon valving was demonstrated to reliably transfer sample liquid in all cases ( n = 35) and CTP two-step aliquoting transfers metered aliquots of 18.2 ± 1.2 μl (CV 6.7%, n = 8) into reaction cavities within 5 s ( n = 24). Thermal characteristics of CTP two-stage aliquoting were found to be in good agreement with an introduced analytical model ( R ~(2) = 0.9876, n = 3). A microfluidic disk segment comprising both new unit operations was used for automation of real-time PCR amplification of Escherichia coli DNA. Required primers and probes were pre-stored in the reaction cavities and a comparison with reference reactions in conventional PCR tubes yielded the same PCR efficiency, repeatability, and reproducibility.
机译：我们将几何多路复用实时PCR的微流体自动化引入了现成的转子 - 基因Q热循环仪（RGQ，Qiagen GmbH，Hilden，Hilden，德国）。对于离心流体控制，RGQ仅提供400 rpm的低且恒定的旋转。通过使用热气体压缩和抗阀的膨胀和等分，实现对离心致动的这种非常有限的离心致动的柔韧性的兼容性。与现有的热气动驱动相比，离心机 - 热气（CTP）流体控制采用全球温度控制作为两个新单元操作的致动参数，采用局部蒸汽压力的诱导变化：CTP虹吸阀和CTP两级等分。证明CTP虹吸阀以可靠地将样品液体转移到所有情况下（n = 35），并在5 s内调位将计量的等分试样为18.2±1.2μl（CV 6.7％，n = 8）的计量等分试样（n = 24）。发现CTP两级等分的热特性与引入的分析模型（R〜（2）= 0.9876，n = 3）吻合良好。包括新单元操作的微流体盘段用于大肠杆菌DNA的实时PCR扩增的自动化。所需的引物和探针预先储存在反应腔中，与常规PCR管中的参考反应的比较产生相同的PCR效率，可重复性和再现性。

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