Compact disk (CD)-shaped device for single cell isolation and PCR of a specific gene in the isolated cell
Compact disk (CD)-shaped device for single cell isolation and PCR of a specific gene in the isolated cell
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DOI：
10.1007/s00216-010-4205-7
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发表时间：
2010-12-01
影响因子：
4.3
通讯作者：
Kubo, Izumi
中科院分区：
文献类型：
--
作者：
Furutani, Shunsuke;Nagai, Hidenori;Kubo, Izumi
为了立即区分分离的细胞，我们提出了一种新颖的设备和技术，用于分离细胞并通过聚合酶链反应（PCR）连续检测其中的特定基因。在本研究中，我们分离了肠道沙门氏菌细胞，并在光盘 (CD) 形装置上通过 PCR 检测了分离细胞中沙门氏菌特异性 invA 基因。该装置无需微型泵即可通过离心力实现液体流动，并由硅片和玻璃制成，以避免少量试剂蒸发。一台设备有 24 个微通道，每个微通道上集成了 313 个微室。将一微升含有细胞的 PCR 混合物以 5000 rpm 的速度分离到装置上的微室中，持续 30 秒。每个微室包含大约 1.5 nL PCR 混合物。对于不同密度的细胞悬浮液，观察到肠沙门氏菌细胞的泊松分布。在200个细胞μL-1的肠沙门氏菌或更少时，可以通过扩增invA基因的DNA在装置上确定分离的单细胞；在 400 个细胞μL-1 的情况下，可以在设备上测定不含、1 个、2 个或 3 个细胞的室。在 CD 形装置上通过 PCR 对肠沙门氏菌和大肠杆菌的混合物进行选择性检测。
For immediate discrimination among isolated cells we propose a novel device and technique for isolation of cells and sequential detection of specific gene(s) within them by polymerase chain reaction (PCR). In this study, we isolated Salmonella enterica cells and detected the Salmonella-specific invA gene from isolated cells by PCR on a compact disk (CD)-shaped device. This device enabled liquid flow by centrifugal force without a micro pump, and was fabricated from silicon wafer and glass to avoid evaporation of a small amount of reagent. One device has 24 microchannels, and 313 microchambers integrated on each microchannel. One microliter of PCR mixture containing cells was separated into microchambers on the device at 5000 rpm for 30 s. Each microchamber contained approximately 1.5 nL PCR mixture. A Poisson distribution of S. enterica cells was observed for different densities of cell suspension. At 200 cells mu L-1 of S. enterica or less, isolated single cells could be determined on the device by amplification of DNA of the invA gene; at 400 cells mu L-1, chambers containing no, one, two, or three cells could be determined on the device. Selective detection of S. enterica was achieved by PCR from a mixture of S. enterica and Esherichia coli on the CD-shaped device.