Digital PCR on a SlipChip.
Digital PCR on a SlipChip.
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DOI：
10.1039/c004521g
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发表时间：
2010-10-21
期刊：
影响因子：
6.1
通讯作者：
Ismagilov RF
中科院分区：
文献类型：
--
作者：
Shen F;Du W;Kreutz JE;Fok A;Ismagilov RF
本文介绍了一种滑动芯片进行数字PCR在一个非常简单和廉价的格式。用于引入与PCR混合物组合的样品的流体路径使用滑动芯片的两个板中的细长威尔斯形成，所述细长孔设计成在样品加载期间重叠。通过两个板的简单滑动来打破该流体路径，从而消除了威尔斯孔之间的重叠，并使每个孔与预加载有油的储层接触，以同时产生1，280个反应室（每个2.6 nL）。热循环后，使用终点荧光强度检测核酸的存在。以金黄色葡萄球菌基因组DNA为模板，对芯片上的数字PCR进行定量检测。随着反应混合物中模板DNA浓度的稀释，阳性威尔斯孔的分数降低，如统计分析所预期。实验期间未观察到交叉污染。在数字PCR动态范围的极端情况下，使用二项分布的正态近似确定的标准置信区间不令人满意。因此，使用基于评分法的统计分析来确定这些置信区间。滑动芯片提供了一种简单的策略，通过使用PCR来计数核酸。它可以在研究应用中找到应用，例如单细胞分析，产前诊断和床旁诊断。滑动芯片将成为诊断的价值，包括在与等温核酸扩增技术和视觉读出集成后在资源有限的领域的应用。
This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1,280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout.
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影响因子：
15
作者：
Li, Liang;Du, Wenbin;Ismagilov, Rustem F.
通讯作者：
Ismagilov, Rustem F.
前列腺癌的基因表达谱揭示了多个分子途径在转移过程中的参与。
影响因子：
3.8
作者：
Chandran, Uma R.;Ma, Changqing;Dhir, Rajiv;Bisceglia, Michelle;Lyons-Weiler, Maureen;Liang, Wenjing;Michalopoulos, George;Becich, Michael;Monzon, Federico A.
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Monzon, Federico A.
影响因子：
1.8
作者：
Agresti, A;Coull, BA
通讯作者：
Coull, BA
影响因子：
3.7
作者：
BLYTH, CR;STILL, HA
通讯作者：
STILL, HA
影响因子：
15
作者：
Li, Liang;Du, Wenbin;Ismagilov, Rustem F.
通讯作者：
Ismagilov, Rustem F.