Real-time PCR of single bacterial cells on an array of adhering droplets
Real-time PCR of single bacterial cells on an array of adhering droplets
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DOI：
10.1039/c1lc20207c
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发表时间：
2011-01-01
期刊：
影响因子：
6.1
通讯作者：
Meldrum, Deirdre R.
中科院分区：
文献类型：
--
作者：
Shi, Xu;Lin, Liang-I;Meldrum, Deirdre R.
单细菌细胞水平的实时荧光PCR是定量揭示同源细胞异质性的重要工具。利用微量滴定板或PCR管的常规PCR平台已被广泛使用，但它们的大反应体积不适合敏感的单细胞分析。微流体装置提供高密度、低体积的PCR室，但是它们通常是昂贵的并且需要专用设备来操纵液体和执行检测。为了解决这些局限性，我们开发了一种廉价的芯片级设备，该设备与商业实时PCR热循环仪兼容，用于对单个细菌细胞进行定量PCR。该芯片包含12个表面粘附的液滴，由亲水性图案定义，当它们浸入油中时，它们作为实时PCR反应室。该方法采用一步法，即在上样前将试剂与细胞培养基预混合，因此不需要芯片上的液体操作和DNA纯化。为了验证其在遗传分析中的应用，将集胞藻PCC 6803细胞从1000个细胞加载到芯片上，每个液滴一个细胞，并在可商购的ABI StepOne实时PCR热循环仪上分析其16 S rRNA基因（每个细胞两个拷贝）。结果表明，该装置能够在单个细菌细胞水平上进行遗传分析，C-q标准偏差小于1.05个循环。在单个细菌细胞水平上，这种基于芯片的操作成功率超过85%。
Real-time PCR at the single bacterial cell level is an indispensable tool to quantitatively reveal the heterogeneity of isogenetic cells. Conventional PCR platforms that utilize microtiter plates or PCR tubes have been widely used, but their large reaction volumes are not suited for sensitive single-cell analysis. Microfluidic devices provide high density, low volume PCR chambers, but they are usually expensive and require dedicated equipment to manipulate liquid and perform detection. To address these limitations, we developed an inexpensive chip-level device that is compatible with a commercial real-time PCR thermal cycler to perform quantitative PCR for single bacterial cells. The chip contains twelve surface-adhering droplets, defined by hydrophilic patterning, that serve as real-time PCR reaction chambers when they are immersed in oil. A one-step process that premixed reagents with cell medium before loading was applied, so no on-chip liquid manipulation and DNA purification were needed. To validate its application for genetic analysis, Synechocystis PCC 6803 cells were loaded on the chip from 1000 cells to one cell per droplet, and their 16S rRNA gene (two copies per cell) was analyzed on a commercially available ABI StepOne real-time PCR thermal cycler. The result showed that the device is capable of genetic analysis at single bacterial cell level with C-q standard deviation less than 1.05 cycles. The successful rate of this chip-based operation is more than 85% at the single bacterial cell level.