Influence of segmenting fluids on efficiency, crossing point and fluorescence level in real time quantitative PCR
Influence of segmenting fluids on efficiency, crossing point and fluorescence level in real time quantitative PCR
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DOI：
10.1007/s10544-006-6383-9
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发表时间：
2006-03-01
影响因子：
2.8
通讯作者：
Gonzalez, A
中科院分区：
文献类型：
--
作者：
Walsh, EJ;King, C;Gonzalez, A
两相分段流动方法在微器件中对生物样品进行处理和定量分析，与单相连续流动方法相比具有显着优势。尽管如此，关于样品和反应物与分段流体的相容性知之甚少，尽管许多研究者已经报道了取决于所采用的分段流体的产率降低和酶促反应的抑制。目前的研究解决了各种分割液与真实的时间定量PCR的兼容性，以了解生物技术中这一重要反应的物理化学要求。结果表明，使用各种分割液，创建静态分割液/PCR混合界面对反应效率、交叉阈值和结束荧光水平的影响可忽略不计。这意味着先前报道的抑制作用是连续流动系统中分段流体和样品之间动态运动的结果。这里介绍的结果是第一步了解的局限性，分段流方法，这是必要的，使这种方法进入主流使用。
The two-phase segmented flow approach to the processing and quantitative analysis of biological samples in microdevices offers significant advantages over the single-phase continuous flow methodology. Despite this, little is known about the compatibility of samples and reactants with segmenting fluids, although a number of investigators have reported reduced yield and inhibition of enzymatic reactions depending on the segmenting fluid employed. The current study addresses the compatibility of various segmenting fluids with real time quantitative PCR to understand the physicochemical requirements of this important reaction in biotechnology. The results demonstrate that creating a static segmenting fluid/PCR mix interface has a negligible impact on the reaction efficiency, crossing threshold and end fluorescence levels using a variety of segmenting fluids. The implication is then that the previously reported inhibitory effects are the result of the dynamic motion between the segmenting fluid and the sample in continuously flowing systems. The results presented here are a first step towards understanding the limitations of the segmented flow methodology, which are necessary to bring this approach into mainstream use.