Real-time PCR of single bacterial cells on an array of adhering droplets.

Abstract

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.