Analytical Sciences

Rapid and Highly Sensitive Detection by a Real-time Polymerase Chain Reaction Using a Chip Coated with Its Reagents

Shunsuke FURUTANI,* Nahoko NARUISHI,* Masato SAITO,** Eiichi TAMIYA,** Yusuke FUCHIWAKI,*** and Hidenori NAGAI*

*Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan
**Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
***Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, Japan

On-site detection by flow-through polymerase chain reaction (PCR) microfluidic systems for rapid and highly sensitive analysis, are significantly desired for bioanalytical and medical research. The conventional continuous-flow PCR chips realized rapid detection, but their sensitivity was very low (10⁶ to 10⁸ copies μL⁻¹). We improved this drawback by coating the chip with a PCR reagents mixture, and succeed to obtain a rapid and highly sensitive detection by using a segment-flow PCR system. In the present work, we developed a portable segment-flow PCR system for practical use. PCR was performed for the uid A gene in E. coli. By real-time segment-flow PCR using coated chips, we realized rapid detection in 8 min and a high sensitivity of 4 cells μL⁻¹. The sensitivity by the segment-flow PCR chip was the same as that of a conventional thermal cycler. Moreover, the detection speed of our segment-flow PCR chip was 15-times as rapid as that of the conventional thermal cycler.

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