Mahmoudian Laili, Kaji Noritada, Tokeshi Manabu, Nilsson Mats, Baba Yoshinobu
Department of Applied Chemistry, Graduate School of Engineering, MEXT Innovative Research Center for Preventive Medical Engineering, and Plasma Nanotechnology Research Center, Nagoya University, Japan.
Anal Chem. 2008 Apr 1;80(7):2483-90. doi: 10.1021/ac702289j. Epub 2008 Feb 29.
We have developed an integrated platform for rolling circle amplification (RCA) and circle-to-circle amplification (C2CA) of circular probe (padlock probe) and subsequent microchip electrophoretic detection of a specific gene on a poly(methyl methacrylate) microchip. RCA and C2CA were successfully carried out at a steady temperature of 37 degrees C in the sample well of the microchip, and their respective product was detected on the same channel of the microchip, which was prefilled with a polymer separation matrix and fluorescent dye. Using a species-specific padlock probe for bacterial pathogen V. cholerae, a 25-ng bacterial genomic DNA could be detected in less than 65 min (including RCA and microchip electrophoresis) by this platform. Stable dsDNA C2CA product of genomic DNA for V. cholerae can be detected with the introduced integrated platform. Furthermore, the usefulness of this technique for the monitoring of RCA was demonstrated. This integrated platform provides a sensitive, fast, high-throughput, and reproducible method for signal amplification and detection of the padlock probes in the same microchip and is a promising tool for highly specific gene detection strategies.
我们开发了一个集成平台，用于对环形探针（锁式探针）进行滚环扩增（RCA）和环到环扩增（C2CA），并随后在聚甲基丙烯酸甲酯微芯片上对特定基因进行微芯片电泳检测。RCA和C2CA在微芯片样品孔中37摄氏度的稳定温度下成功进行，它们各自的产物在预先填充有聚合物分离基质和荧光染料的微芯片同一通道上被检测到。使用针对细菌病原体霍乱弧菌的物种特异性锁式探针，通过该平台可在不到65分钟内（包括RCA和微芯片电泳）检测到25纳克细菌基因组DNA。利用所引入的集成平台可以检测到霍乱弧菌基因组DNA的稳定双链DNA C2CA产物。此外，还证明了该技术对RCA监测的实用性。这个集成平台为在同一微芯片中对锁式探针进行信号放大和检测提供了一种灵敏、快速、高通量且可重复的方法，是用于高特异性基因检测策略的一个有前景的工具。
